Friday, January 25, 2008

Susceptibility of Various Genotypic Strains to Pyrimethamine, Sulfadiazine and Atovaquone

Antimicrob Agents Chemother. 2008 Jan 22 [Epub ahead of print]

Toxoplasma gondii: In Vitro Susceptibility of Various Genotypic Strains to Pyrimethamine, Sulfadiazine and Atovaquone

Meneceur P, Bouldouyre MA, Aubert D, Villena I, Menotti J, Sauvage V, Garin JF, Derouin F.

Laboratory of Parasitology (EA 3520), University Denis Diderot, 15 rue de l'école de médecine, 75250 Paris cédex 06, France; Hôpital Saint-Louis, Assistance Publique Hôpitaux de Paris, 1, avenue Claude Vellefaux, 75010, Paris, France; Laboratory of Parasitology (EA 3800), and Biological Resource Centre Toxoplasma; University of Reims Champagne-Ardennes and Hôpital Maison-Blanche, 45 rue Cognacq-Jay, 51092 Reims cédex, France.

Sulfadiazine, pyrimethamine and atovaquone are widely used for treatment of severe toxoplasmosis. Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I. We determined the in vitro activities of these drugs against 17 strains of T. gondii belonging to various genotypes and examined the correlations between IC50's, growth kinetics, strain genotype and mutations on drug target genes. Growth kinetics was determined in THP-1 cell cultures using real-time PCR. IC50's were determined in MRC-5 cell cultures using a T. gondii specific ELISA performed on cultures. Mutations in DHFR, DHPS and Cytochrome b genes were looked for by sequencing. Pyrimethamine IC50's ranged between 0.07 and 0.39 mg/L with no correlation with the strain genotype but a significant correlation with growth kinetics. Several mutations found on DHFR gene were not linked to a lower susceptibility. Atovaquone IC50's were in a narrow range of concentrations (mean 0.06+/-0.02 mg/L); no mutation was found on the Cytochrome b gene. IC50's for sulfadiazine ranged between 3 and 18.9 mg/L for 13 strains and were >50 mg/L for 3 strains. High IC50's were not correlated to strain genotypes or growth kinetics. A new mutation of the DHPS gene was demonstrated in one of these strains. In conclusion, we found variability in the susceptibility of T. gondii strains to pyrimethamine and atovaquone with no evidence of drug resistance. A higher variability was found for sulfadiazine with a possible resistance for 3 strains. No relationship was found between drug susceptibility and strain genotype.

PMID: 18212105 [PubMed - as supplied by publisher]

Crystal structures of Toxoplasma gondii pterin-4a-carbinolamine dehydratase

Mol Biochem Parasitol. 2007 Dec 15 [Epub ahead of print]

Crystal structures of Toxoplasma gondii pterin-4a-carbinolamine dehydratase and comparisons with mammalian and parasite orthologues

Cameron S, Fyffe SA, Goldie S, Hunter WN.

Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

The enzyme pterin-4a-carbinolamine dehydratase (PCD) is important for the recycling of pterins within eukaryotic cells. A recombinant expression system for PCD from the apicomplexan parasite Toxoplasma gondii has been prepared, the protein purified and crystallised. Single crystal X-ray diffraction methods have produced a high-resolution structure (1.6A) of the apo-enzyme and a low-resolution structure (3.1A) of a complex with a substrate-like ligand dihydrobiopterin (BH(2)). Analysis of the hydrogen bonding interactions that contribute to binding BH(2) suggest that the ligand is present in an enol tautomeric state, which makes it more similar to the physiological substrate. The enzyme can process (R)- and (S)-forms of pterin-4a-carbinolamine and the ligand complex suggests that His61 and His79 are placed to act independently as general bases for catalysis of the individual enantiomers. Comparisons with orthologues from other protozoan parasites (Plasmodium falciparum and Leishmania major) and with rat PCD, for which the structure is known, indicate a high degree of sequence and structure conservation of this enzyme. The molecular determinants of ligand recognition and PCD reactivity are therefore highly conserved across species.

PMID: 18215430 [PubMed - as supplied by publisher]

Wednesday, January 23, 2008

Organizational Changes of the Daughter Basal Complex during the Parasite Replication of Toxo

PLoS Pathog. 2008 Jan 18;4(1):e10 [Epub ahead of print]

Organizational Changes of the Daughter Basal Complex during the Parasite Replication of Toxoplasma gondii

Hu K.

The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

PMID: 18208326 [PubMed - as supplied by publisher]

Tuesday, January 22, 2008

Enzyme-linked immunosorbent assay using different fragments of MIC1 for detection of immunoglobulin G antibodies

Exp Parasitol. 2008 Jan 18 [Epub ahead of print]

Toxoplasma gondii: Enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies

Holec L, Gąsior A, Brillowska-Dąbrowska A, Kur J

Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.

Three different fragments of microneme 1 protein termed, r-MIC1ex2, r-MIC1ex34 and r-MIC1 of Toxoplasma gondii, were expressed in Escherichia coli as fusion proteins containing six histidyl residues at N- and C-terminal. After purification by metal affinity chromatography, these recombinant proteins were tested for their usefulness as antigens in an enzyme-linked immunosorbent assay for the detection of immunoglobulin G. Ninety-eight sera from patients with different stages of invasion and 24 sera from seronegative patients were examined. There was no significant difference observed in the antigenicity for human serum samples from patients with acute toxoplasmosis between three recombinant types of MIC1 antigen (96.1% for r-MIC1ex2 antigen and 100% for both r-MIC1ex34 and r-MIC1 proteins). Sera from chronic infections (with low titers of IgG antibody) showed significant lower sensitivity, especially for r-MIC1ex34 and r-MIC1 antigens (75%, 52.7% and 36.1% for r-MIC1ex2, r-MIC1ex34 and r-MIC1, respectively). These results indicate that the strongest antigenic region of the MIC1 is encoding by the second exon of mic1 gene. When r-MIC1ex2 (N-terminal fragment of protein) was combined with MAG1 (matrix antigen) and MIC3 (microneme 3 protein), the sensitivity increased to 88.9%. This result was comparable to an ELISA using a Toxoplasma lysate antigen (TLA) and two combinations of recombinant antigens: M1 (GRA1+GRA7+SAG1) and M2 (P35+SAG2+GRA6) with the sensitivity for serum samples tested 94.4%, 88.9% and 94.4%, respectively.

PMID: 18207143 [PubMed - as supplied by publisher]

Sex-dependent toxoplasmosis-associated differences in testosterone concentration in humans

Parasitology. 2008 Jan 21;:1-5 [Epub ahead of print]

Sex-dependent toxoplasmosis-associated differences in testosterone concentration in humans

Flegr J, Lindová J, Kodym P.

Department of Parasitology, Faculty of Sciences, Charles University, Viničná 7, 128 44 Prague 2, Czech Republic.

SUMMARYSeveral lines of indirect evidence suggest that subjects with latent infection of the coccidian parasite Toxoplasma gondii have a higher concentration of testosterone than uninfected controls. Here, we searched for direct evidence of latent toxoplasmosis-associated differences in testosterone concentration among a population of 174 female and 91 male students screened for Toxoplasma infection. We have found Toxoplasma-infected men to have a higher concentration of testosterone and Toxoplasma-infected women to have a lower concentration of testosterone than Toxoplasma-free controls. The opposite direction of the testosterone shift in men compared to women can explain the observed gender specificity of behavioural shifts in Toxoplasma-infected subjects.

PMID: 18205984 [PubMed - as supplied by publisher]

Role of IFN-gamma-induced tryptophan degradation

FEMS Immunol Med Microbiol. 2008 Jan 16 [Epub ahead of print]

Antimicrobial and immunoregulatory effects mediated by human lung cells: role of IFN-gamma-induced tryptophan degradation

Heseler K, Spekker K, Schmidt SK, Mackenzie CR, Däubener W

Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-Universität Düsseldorf, Germany.

Pneumonia caused by bacterial, viral and parasitic pathogens is one of the most common clinical problems facing primary and secondary care physicians. Staphylococcus aureus is a common cause of lung abscesses in humans and, in immunocompromised patients, herpes simplex virus type I and Toxoplasma gondii can cause severe life-threatening pneumonia. The authors focused their interest in the antimicrobial effects mediated by human lung cells against these pathogens. It was found that IFN-gamma-stimulated lung cells are capable of inhibiting T cell proliferation and restrict the replication of microorganisms such as T. gondii, S. aureus and herpes simplex virus. This immunoregulatory and antimicrobial effect was enhanced in the presence of IL-1 or tumor necrosis factor-alpha (TNF-alpha). Furthermore, the IFN-gamma-dependent antimicrobial effects of HBE4-E6/E7 (human lung bronchus epithelial cells) and A549 (human type II alveolar cells) correlated with the activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). It was found that both the abrogation of IDO activity by the specific IDO-inhibitor 1-l-methyltryptophan and the supplementation of cultures with tryptophan result in an inhibition of IFN-gamma-induced antimicrobial effects mediated by lung cells. Therefore it is suggested that tryptophan depletion via IFN-gamma-mediated IDO induction is a major antibacterial, antiparasitic, antiviral and immunoregulatory mechanism in human lung cells.

PMID: 18205804 [PubMed - as supplied by publisher]

MARveling at parasite invasion

Trends Parasitol. 2008 Jan 17 [Epub ahead of print]

MARveling at parasite invasion

Hager KM, Carruthers VB

Department of Biological Sciences, Center for Global Health and Infectious Disease, University of Notre Dame, Notre Dame, IN 46556-0369, USA.

Micronemal proteins (MICs) are key mediators of cytoadherence and invasion for Toxoplasma gondii. Emerging evidence indicates that carbohydrate binding facilitates Toxoplasma entry into host cells. The recently solved Toxoplasma MIC1s (TgMIC1s) structure reveals the presence of novel specialized domains that can discriminate between glycan residues. Comparison with Plasmodium erythrocyte-binding antigen 175 reveals that terminal sialic acid residues might represent a shared but tailored invasion pathway among apicomplexan parasites.

PMID: 18203663 [PubMed - as supplied by publisher]

Saturday, January 19, 2008

'Golden Bullet' Shows Promise For Killing Common Parasite

"Researchers in Australia report development of a new type of gold nanoparticle that destroys the parasite responsible for toxoplasmosis, a potentially serious disease acquired by handling the feces of infected cats or eating undercooked meat. Their so-called "golden bullet" could provide a safer, more effective alternative for treating the disease than conventional drug therapy, they say..."

Read story.

Toxoplasma Infection Increases Risk of Schizophrenia

"Findings from what is believed to be the largest comparison of blood samples collected from healthy individuals and people with schizophrenia suggest that infection with the common Toxoplasma gondii parasite, carried by cats and farm animals, may increase the risk of schizophrenia..."

Story here.

ProtozoaDB: dynamic visualization and exploration of protozoan genomes

Nucleic Acids Res. 2008 Jan;36(Database issue):D547-52. Epub 2007 Nov 2

ProtozoaDB: dynamic visualization and exploration of protozoan genomes

Dávila AM, Mendes PN, Wagner G, Tschoeke DA, Cuadrat RR, Liberman F, Matos L, Satake T, Ocaña KA, Triana O, Cruz SM, Jucá HC, Cury JC, Silva FN, Geronimo GA, Ruiz M, Ruback E, Silva FP Jr, Probst CM, Grisard EC, Krieger MA, Goldenberg S, Cavalcanti MC, Moraes MO, Campos ML, Mattoso M.

Oswaldo Cruz Institute, FIOCRUZ, CCB, Federal University of Santa Catarina (UFSC), ACBS/UNOESC/SC, COPPE, Federal University of Rio de Janeiro, Brazil.

ProtozoaDB ( is being developed to initially host both genomics and post-genomics data from Plasmodium falciparum, Entamoeba histolytica, Trypanosoma brucei, T. cruzi and Leishmania major, but will hopefully host other protozoan species as more genomes are sequenced. It is based on the Genomics Unified Schema and offers a modern Web-based interface for user-friendly data visualization and exploration. This database is not intended to duplicate other similar efforts such as GeneDB, PlasmoDB, TcruziDB or even TDRtargets, but to be complementary by providing further analyses with emphasis on distant similarities (HMM-based) and phylogeny-based annotations including orthology analysis. ProtozoaDB will be progressively linked to the above-mentioned databases, focusing in performing a multi-source dynamic combination of information through advanced interoperable Web tools such as Web services. Also, to provide Web services will allow third-party software to retrieve and use data from ProtozoaDB in automated pipelines (workflows) or other interoperable Web technologies, promoting better information reuse and integration. We also expect ProtozoaDB to catalyze the development of local and regional bioinformatics capabilities (research and training), and therefore promote/enhance scientific advancement in developing countries.

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

PMID: 17981844 [PubMed - in process]

An emerging cyberinfrastructure for biodefense pathogen and pathogen-host data

Nucleic Acids Res. 2008 Jan;36(Database issue):D884-91. Epub 2007 Nov 4

An emerging cyberinfrastructure for biodefense pathogen and pathogen-host data

Zhang C, Crasta O, Cammer S, Will R, Kenyon R, Sullivan D, Yu Q, Sun W, Jha R, Liu D, Xue T, Zhang Y, Moore M, McGarvey P, Huang H, Chen Y, Zhang J, Mazumder R, Wu C, Sobral B.

Virginia Bioinformatics Institute at Virginia Polytechnic Institute and State University, Washington Street (0477), Blacksburg, VA 24061, USA.

The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host-pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at

Publication Types:
Research Support, N.I.H., Extramural

PMID: 17984082 [PubMed - in process]

Wednesday, January 16, 2008

Advances in the use of genetically engineered parasites to study immunity to Toxo

Parasite Immunol. 2008 Jan 8 [Epub ahead of print]

Advances in the use of genetically engineered parasites to study immunity to Toxoplasma gondii

Dzierszinski FS, Hunter CA

Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Canada

Studying in vivo biology and the host immune response to Toxoplasma gondii has yielded many insights into the pathogenesis of this parasitic organism. It is recognized that this infection in immune competent hosts elicits a strong Th1-type response, which is characterized by the generation of parasite-specific CD4(+) and CD8(+) T cells that produce IFN-gamma and provide protective immunity. One of the problems associated with studying resistance to Toxoplasma has been the lack of reagents to track parasite-specific T cell responses with a high degree of specificity. To overcome this difficulty, it is possible to use a combination of transgenic parasites that are engineered to express well-characterized heterologous reporters or antigens, and T cell hybridomas or naïve T cells that express a T cell receptor specific for the processed peptide. These approaches have provided new insights into parasite dissemination, antigen presentation, as well as immune regulation.

PMID: 18194347 [PubMed - as supplied by publisher]

Breakthrough cerebral toxoplasmosis in a patient receiving atovaquone prophylaxis

Pediatr Transplant. 2008 Jan 8 [Epub ahead of print]

Breakthrough cerebral toxoplasmosis in a patient receiving atovaquone prophylaxis after a hematopoietic stem cell transplantation

Megged O, Shalit I, Yaniv I, Stein J, Fisher S, Levy I.

Infectious Diseases Unit, Schneider Children's Medical Center of Israel, Petah Tiqwa, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

We describe a case of breakthrough cerebral toxoplasmosis during atovaquone therapy in a child who was intolerant of conventional prophylactic regimens after hematopoietic stem cell transplantation. The available data on the efficacy of atovaquone prophylaxis in Toxoplasma sero-positive stem cell transplant recipients remain limited, and other strategies, such as preemptive strategy using toxoplasma PCR or TMP-SMX desensitization should be considered in this setting.

PMID: 18194354 [PubMed - as supplied by publisher]

Migration of Apicomplexa Across Biological Barriers: the Toxoplasma and Plasmodium rides

Traffic. 2008 Jan 9 [Epub ahead of print]

Migration of Apicomplexa Across Biological Barriers: the Toxoplasma and Plasmodium rides

Tardieux I, Ménard R.

Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique UMR 8104; and Institut National de la Santé et de la Recherche Médicale U567, Paris, France.

The invasive stages of Apicomplexa parasites, called zoites, have been largely studied in in vitro systems, with a special emphasis on their unique gliding and host cell invasive capacities. In contrast, the means by which these parasites reach their destination in their hosts are still poorly understood. We summarize here our current understanding of the cellular basis of in vivo parasitism by two well-studied Apicomplexa zoites, the Toxoplasma tachyzoite and the Plasmodium sporozoite. Despite being close relatives, these two zoites use different strategies to reach their goal and establish infection.

PMID: 18194412 [PubMed - as supplied by publisher]

Co-infection with Heligmosomoides polygyrus fails to establish a CD8+ T cell immunity against Toxo

Infect Immun. 2008 Jan 14 [Epub ahead of print]

Co-infection with Heligmosomoides polygyrus fails to establish a CD8+ T cell immunity against Toxoplasma gondii

Khan IA, Hakak R, Eberle K, Sayles P, Weiss LM, Urban JF Jr.

Department of Microbiology and Tropical Medicine and Immunology George Washington University, Washington DC, Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, Trudeau Institute Saranac Lake, New York, Department of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, New York and USDA/ARS/Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, Beltsville, Maryland.

CD8(+) T cell immunity is important for long-term protection against Toxoplasma gondii infection. However, a Th1 cytokine environment, especially the presence of IFNgamma, is essential for the development of primary CD8(+) T cell immunity against this obligate intracellular pathogen. Earlier studies from our laboratory have demonstrated that mice lacking optimal IFNgamma levels fail to develop robust CD8(+) T cell immunity against T. gondii. In the present study, induction of primary CD8(+) T cell immune response against T. gondii infection was evaluated in mice infected earlier with Heligmosomoides polygyrus, a gastrointestinal worm known to evoke a polarized Th2 response in the host. In the early stage of T. gondii infection both CD4 and CD8(+) T cell responses against the parasite were suppressed in the dual infected mice. At the later stages, however, T. gondii-specific CD4(+) T cell immunity recovered, while CD8(+) T cell responses remained low. Unlike mice infected with T. gondii alone, depletion of CD4(+) T cells in the dual infected mice led to reactivation of chronic infection, leading to Toxoplasma-related encephalitis. Our observations strongly suggest that prior infection with a Th2 cytokine polarizing pathogen can inhibit the development of CD8(+) T cell immune response against T. gondii, thus compromising long-term protection against a protozoan parasite. This is the first study to examine the generation of CD8(+) T cell immune response in a parasitic nematode and protozoan co-infection model that has important implications for infections where a CD8(+) T cell response is critical for host protection and reduced infection pathology.

PMID: 18195022 [PubMed - as supplied by publisher]

Tuesday, January 15, 2008

High prevalence and abundant atypical genotypes of Toxo isolated from lambs

Int J Parasitol. 2007 Dec 8 [Epub ahead of print]

High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA

Dubey JP, Sundar N, Hill D, Velmurugan GV, Bandini LA, Kwok OC, Majumdar D, Su C.

United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA.

Little information is available on the presence of viable Toxoplasma gondii in tissues of lambs worldwide. The prevalence of T. gondii was determined in 383 lambs (<1 year old) from Maryland, Virginia and West Virginia, USA. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100 and 1:200 dilutions, and hearts were selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. For bioassays in cats, the entire myocardium or 500g was chopped and fed to cats, one cat per heart and faeces of the recipient cats were examined for shedding of T. gondii oocysts. For bioassays in mice, 50g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. In total, 53 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 53 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed 57 strains with 15 genotypes. Four lambs had infections with two T. gondii genotypes. Twenty-six (45.6%) strains belong to the clonal Type II lineage (these strains can be further divided into two groups based on alleles at locus Apico). Eight (15.7%) strains belong to the Type III lineage. The remaining 22 strains were divided into 11 atypical genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in lambs, which has important implications in public health. We believe this is the first in-depth genetic analysis of T. gondii isolates from sheep in the USA.

PMID: 18191859 [PubMed - as supplied by publisher]

Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection

Immunol Res. 2008;40(1):35-48

Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection

Egan CE, Sukhumavasi W, Bierly AL, Denkers EY.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853-6401, USA,

The murine cell surface determinant Gr-1 is expressed at high level on neutrophils. Depletion of polymorphonuclear leukocytes with anti-Gr-1(+) monoclonal antibody results in increased susceptibility and dysregulated immunity to many microbial pathogens, a finding widely interpreted to indicate the importance of neutrophils during infection. Yet, in recent years it has become clear that additional cell types express the Gr-1 determinant, including dendritic cell and monocyte subpopulations. In this review, we evaluate current knowledge on the functional aspects of Gr-1(+) cell populations. We focus on infection with the opportunistic protozoan Toxoplasma gondii, a case where host survival depends on an intact Gr-1(+) cell population.

PMID: 18193362 [PubMed - in process]

Sunday, January 13, 2008

Transplacental toxoplasmosis in naturally-infected white-tailed deer

Int J Parasitol. 2007 Dec 5 [Epub ahead of print]

Transplacental toxoplasmosis in naturally-infected white-tailed deer: Isolation and genetic characterisation of Toxoplasma gondii from foetuses of different gestational ages.

Dubey JP, Velmurugan GV, Ulrich V, Gill J, Carstensen M, Sundar N, Kwok OC, Thulliez P, Majumdar D, Su C.

United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA.

Clinical toxoplasmosis is most severe in congenitally-infected hosts. In humans, transmission of Toxoplasma gondii from the mother to the foetus is considered to be most efficient during the last trimester of pregnancy but clinical congenital toxoplasmosis is more severe if transmission occurs during the first trimester. However, there are no data on the rate of congenital transmission of T. gondii with respect to gestational age in any host during natural infection. In the present study, attempts were made to isolate T. gondii by bioassay in mice inoculated with tissues from foetuses of 88 naturally-exposed white-tailed deer from Iowa and Minnesota. Viable T. gondii was isolated from foetuses of six of 61 deer in early pregnancy (45-85 days of gestation) from Iowa and foetuses of nine of 27 deer from Minnesota in mid-gestation (130-150 days) of a gestational period of 7 months. The 15 T. gondii isolates obtained from foetal deer were PCR-restriction fragment length polymorphism genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and an apicoplast marker, Apico. Five genotypes were revealed, including the clonal Type II and III lineages, and three non-clonal genotypes. DNA sequencing analysis of representative isolates at loci SAG2, c22-8, L358 and PK1 revealed that the three non-clonal genotypes are closely related to the clonal Type I, II and III lineages. It is very likely that these non-clonal genotypes were derived from genetic crosses among the three clonal Type I, II and III lineages. The most common genotype was Type II, commonly found in humans in North America and Europe, suggesting the possible link of transmission from game animals to humans.

PMID: 18187136 [PubMed - as supplied by publisher]

Proteomic and glycomic analyses of N-glycosylated structures involved in toxo-host cell interactions

Mol Cell Proteomics. 2008 Jan 9 [Epub ahead of print]

Proteomic and glycomic analyses of N-glycosylated structures involved in toxoplasma gondii-host cell interactions

Fauquenoy S, Morelle W, Hovasse A, Bednarczyk A, Slomianny C, Schaeffer C, Van Dorsselaer A, Tomavo S.

Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq 59655.

The apicomplexan parasite Toxoplasma gondii recognizes, binds and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here, we report comprehensive proteomic and glycomic analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man5-9GlcNAc2) and paucimannosidic (Man3-4GlcNAc2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using Concanavalin A (Con A) and Pisum sativum agglutinin (PSA) predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomic and glycan analyses identified components involved in gliding motility, moving junction and other additional functions implicated in intracellular development. Importantly, tunicamycin-treated parasites are considerably reduced in motility, host cell invasion and growth. Collectively, these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

PMID: 18187410 [PubMed - as supplied by publisher]

Toxoplasma gondii and Toxocara spp. Co-infection

Am J Trop Med Hyg. 2008 Jan;78(1):35-39.

Toxoplasma gondii and Toxocara spp. Co-infection

Jones JL, Kruszon-Moran D, Won K, Wilson M, Schantz PM.

Division of Parasitic Diseases, National Center for Zoonotic, Vectorborne, and Enteric Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Division of Health and Nutrition Examination Statistics, National Center for Health Statistics, Centers for Disease Control and Prevention, Hyattsville, Maryland.

Toxoplasma gondii and Toxocara spp. infections can cause systemic and ocular disease. To estimate the prevalence of infection with these organisms, we tested serum samples from persons >/= 12 years of age obtained in the Third National Health and Nutrition Examination Survey (1988-1994). Among those tested for both T. gondii and Toxocara spp. (n = 16,646), the age-adjusted T. gondii antibody prevalence was 23.6% (95% confidence limit [CL] = 22.1-25.1%) and the Toxocara spp. antibody prevalence was 14.0% (95% CL = 12.7-15.4%). Multivariate analysis controlling demographic and risk factors showed that persons infected with Toxocara spp. were more likely to be infected with T. gondii (odds ratio [OR] = 1.93, 95% CL = 1.61-2.31), and similarly, persons infected with T. gondii were more likely to be infected with Toxocara spp. (OR = 1.91, 95% CL = 1.59-2.28). Infection with T. gondii and Toxocara spp. are common and can be prevented by many similar interventions.

PMID: 18187782 [PubMed - as supplied by publisher]

Friday, January 11, 2008

Abscisic acid controls calcium-dependent egress and development in Toxo

Nature. 2008 Jan 10;451(7175):207-10

Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii

Nagamune K, Hicks LM, Fux B, Brossier F, Chini EN, Sibley LD

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

Calcium controls a number of critical events, including motility, secretion, cell invasion and egress by apicomplexan parasites. Compared to animal and plant cells, the molecular mechanisms that govern calcium signalling in parasites are poorly understood. Here we show that the production of the phytohormone abscisic acid (ABA) controls calcium signalling within the apicomplexan parasite Toxoplasma gondii, an opportunistic human pathogen. In plants, ABA controls a number of important events, including environmental stress responses, embryo development and seed dormancy. ABA induces production of the second-messenger cyclic ADP ribose (cADPR), which controls release of intracellular calcium stores in plants. cADPR also controls intracellular calcium release in the protozoan parasite T. gondii; however, previous studies have not revealed the molecular basis of this pathway. We found that addition of exogenous ABA induced formation of cADPR in T. gondii, stimulated calcium-dependent protein secretion, and induced parasite egress from the infected host cell in a density-dependent manner. Production of endogenous ABA within the parasite was confirmed by purification (using high-performance liquid chromatography) and analysis (by gas chromatography-mass spectrometry). Selective disruption of ABA synthesis by the inhibitor fluridone delayed egress and induced development of the slow-growing, dormant cyst stage of the parasite. Thus, ABA-mediated calcium signalling controls the decision between lytic and chronic stage growth, a developmental switch that is central in pathogenesis and transmission. The pathway for ABA production was probably acquired with an algal endosymbiont that was retained as a non-photosynthetic plastid known as the apicoplast. The plant-like nature of this pathway may be exploited therapeutically, as shown by the ability of a specific inhibitor of ABA synthesis to prevent toxoplasmosis in the mouse model.

Subcellular localization and dynamics of a digalactolipid-like eitope in Toxo

J Lipid Res. 2008 Jan 8 [Epub ahead of print]

Subcellular localization and dynamics of a digalactolipid-like eitope in Toxoplasma gondii

Botte C, Saidani N, Mondragon R, Mondragon M, Isaac G, Mui E, McLeod R, Dubremetz JF, Vial H, Welti R, Cesbron-Delauw MF, Mercier C, Marechal E

Toxoplasma gondii is a unicellular parasite characterized by a unique extracellular and intracellular membrane compartments. Lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. Occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE), Immunolabelling characterization supports that DGLE polar head is similar to that of DGDG. Mass spectrometry analyses pointed dihexosyl-lipids having various hydrophobic moieties (ceramide, diacylglycerol, acylalkylglycerol), that might react with anti-DGDG, but we cannot exclude that more complex dihexosyl-terminated lipids might also be immunolabelled. DGLE localization was analyzed by immunofluorescent and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE is scattered in pellicle membrane domains in extracellular tachyzoites and is relocalized to the anterior tip of the cell upon invasion, in an actin-dependent manner, providing insights on a possible role in pathogenetic process. DGLE was detected in other Apicomplexa, i.e. Neospora, Plasmodium, Babesia and Cryptosporidium.

PMID: 18182683 [PubMed - as supplied by publisher]

Infection with Toxo results in dysregulation of the host cell cycle

Cell Microbiol. 2008 Jan 7 [Epub ahead of print]

Infection with Toxoplasma gondii results in dysregulation of the host cell cycle

Molestina RE, El-Guendy N, Sinai AP

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, 40536 USA.

Mammalian cells infected with Toxoplasma gondii are characterized by a profound reprogramming of gene expression. We examined whether such transcriptional responses were linked to changes in the cell cycle of the host. Human foreskin fibroblasts (HFF) in the G(0)/G(1) phase of the cell cycle were infected with T. gondii and FACS analysis of DNA content was performed. Cell cycle profiles revealed a promotion into the S phase followed by an arrest towards the G(2)/M boundary with infection. This response was markedly different from that of growth factor stimulation which caused cell cycle entry and completion. Transcriptional profiles of T. gondii-infected HFF showed sustained increases in transcripts associated with a G(1)/S transition and DNA synthesis coupled to an abrogation of cell cycle regulators critical in G(2)/M transition relative to growth factor stimulation. These divergent responses correlated with a distinct temporal modulation of the critical cell cycle regulator kinase ERK by infection. While the kinetics of ERK phosphorylation by EGF showed rapid and sustained activation, infected cells displayed an oscillatory pattern of activation. Our results suggest that T. gondii infection induces and maintains a "proliferation response" in the infected cell which may fulfill critical growth requirements of the parasite during intracellular residence.

PMID: 18182087 [PubMed - as supplied by publisher]

Thursday, January 10, 2008

Blocking Parasites' Communication Reduces Infection

Blocking Parasites' Communication Reduces Infection

One of the most common human parasites, Toxoplasma gondii, uses a hormone lifted from the plant world to decide when to increase its numbers and when to remain dormant, researchers at Washington University School of Medicine in St. Louis have found.

The scientists report this week in Nature that they successfully blocked production of the molecule, known as abscisic acid (ABA), with a plant herbicide. Low doses of the herbicide prevented fatal T. gondii infection in mice.

Story here.

Tuesday, January 08, 2008

Toxo and Cryptosporidium parvum lack detectable DNA cytosine methylation

Eukaryot Cell. 2008 Jan 4 [Epub ahead of print]

Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation.

Gissot M, Choi SW, Thompson RF, Greally JM, Kim K.

Departments of Medicine (Infectious Diseases) and Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Jean Mayer USDA Human Nutrition Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA.; Departments of Medicine (Hematology) and Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Epigenetic factors play a role in expression of virulence traits in Apicomplexa. Apicomplexan genomes encode putative DNA cytosine methylation enzymes. To assess the presence of cytosine methylation of T.gondii and C. parvum DNA, we used mass spectrometry analysis and confirmed that these organisms lack detectable methylcytosine in their DNA.

PMID: 18178772 [PubMed - as supplied by publisher]

Sunday, January 06, 2008

Toxoplasma gondii ferredoxin-NADP(+) reductase

Proteins. 2008 Jan 3 [Epub ahead of print]

Toxoplasma gondii ferredoxin-NADP(+) reductase: Role of ionic interactions in stabilization of native conformation and structural cooperativity.

Singh K, Bhakuni V.

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

The apicoplast and the proteins present therein are parasite-specific targets for chemotherapy of apicomplexan parasites. Ferredoxin-NADP(+) reductase (FNR) is an important enzyme present in the apicoplast of Toxoplasma gondii that operates as a general electron switch at the bifurcation step of many different electron transfer pathways. In spite of its importance as drug target not much structural information on the enzyme is available. Using fluorescence and CD spectroscopy in combination with enzyme activity measurement and size exclusion chromatography, we studied the pH-dependent changes in structural and functional properties and interdomain interactions in recombinant Toxoplasma gondii ferredoxin-NADP(+) reductase (TgFNR) to understand the interactions responsible for stabilization of native conformation and modulation of functional activity of the enzyme. Under physiological conditions, the recombinant TgFNR is stabilized in an open conformation. The open conformation of the enzyme was found to be essential for its optimum functioning, as induction of compactness/rigidity by modulation of pH, leads to decrease in the functional activity. In native conformation, strong interactions exist between the NADP(+)- and FAD-binding domains thus making the enzyme a structurally cooperative molecule. Under acidic conditions (pH about 4), the interdomain interactions present in native TgFNR were lost and the enzyme became structurally noncooperative. The pH-induced structural alterations in the NADP(+) binding domain, more precisely compaction of the conformation lead to its stabilization against thermal denaturation. The studies demonstrate the significance of electrostatic interactions both in stabilization of native conformation and maintenance of structural cooperativity in TgFNR. Proteins 2008. (c) 2007 Wiley-Liss, Inc.

PMID: 18175327 [PubMed - as supplied by publisher]

Early and Accurate Diagnosis of Congenital Toxoplasmosis

Pediatr Infect Dis J. 2008 Jan 2 [Epub ahead of print]

Early and Accurate Diagnosis of Congenital Toxoplasmosis.

Ciardelli L, Meroni V, Avanzini MA, Bollani L, Tinelli C, Garofoli F, Gasparoni A, Stronati M.

From the *Research Laboratories (Neonatal Immunology and Paediatric Oncohematology), †Infectious Diseases Department, University of Pavia, ‡Neonatal Intensive Care Unit, and §Biometric Unit, Fondazione IRCCS Policlinico San Matteo, Pavia; and ∥Neonatal Intensive Care Unit, Spedali Civili, Brescia, Italy.

OBJECTIVE:: Early diagnosis of congenital toxoplasma infection is difficult to establish using serological methods. We explored specific T cell immunity to Toxoplasma gondii antigens to identify more accurate diagnostic tests for an early diagnosis of toxoplasma infection in newborns at risk for congenital toxoplasmosis. STUDY DESIGN:: T lymphocyte proliferation, interferon (IFN)-gamma production and lymphocyte activation antigens expression were evaluated in 23 infected and 65 uninfected neonates at different times, in the first year of life. RESULTS:: The immunologic tests accurately discriminated when tested 90 days of age, respectively and were significantly lower in uninfected than in infected infants: activation antigen CD25, P < 0.001 and P < 0.00001; activation antigen histocompatibility leukocyte antigen (HLA)-DR, P < 0.01 and P < 0.00001; T cell proliferation, P < 0.0001 and P < 0.00001; IFN-gamma production, P < 0.001 and P < 0.00001. Evaluation of the specific T cell response allowed identification at 3 months of age or younger, 2 of 23 infected neonates, who had negative serologic tests. Moreover specific T lymphocyte activity increased with age even in neonates undergoing therapy, suggesting that medical treatment does not affect lymphocyte response. CONCLUSIONS:: Evaluation of T cell immunity is important for an early and accurate diagnosis of congenital toxoplasmosis.

PMID: 18174870 [PubMed - as supplied by publisher]

Tuesday, January 01, 2008

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic

J Parasitol. 2007 Oct;93(5):1216-8

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic
Bartova E, Sedlak K, Pavlik I, Literak I

Department of Biology and Wildlife Diseases, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.

PMID: 18163361 [PubMed - in process]

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

J Parasitol. 2007 Oct;93(5):1033-5.

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Dubey JP, Alvarado-Esquivel C, Liesenfeld O, Herrera-Flores RG, Ramírez-Sánchez BE, González-Herrera A, Martínez-García SA, Bandini LA, Kwok OC

United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Beltsville, Maryland 20705-2350, USA.

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.

PMID: 18163336 [PubMed - in process]

Seroprevalence of Toxoplasma gondii antibodies in cats from Durango City, Mexico

J Parasitol. 2007 Oct;93(5):1214-6

Seroprevalence of Toxoplasma gondii antibodies in cats from Durango City, Mexico

Alvarado-Esquivel C, Liesenfeld O, Herrera-Flores RG, Ramírez-Sánchez BE, González-Herrera A, Martínez-García SA, Dubey JP

Facultad de Medicina, Universidad Juárez del Estado de Durango, Dgo Mexico.

The prevalence of antibodies to Toxoplasma gondii was determined in sera from 105 domestic cats from Durango City, Mexico. Using a modified agglutination test, antibodies to this parasite were found in 21% of the 105 cats, with titers of 1:25 in 3 cats, 1:50 in 4 cats, 1:200 in 5 cats, 1:400 in 2 cats, 1:800 in 2 cats, 1:1,600 in 4 cats, and 1:3,200 or higher in 2 cats. Cats older than 1 yr had a significantly higher frequency of infection than that found in cats younger than 0.5 yr (41 vs. 13.2%, respectively; odds ratio = 4.55; 95% CI = 1.24-17.18; P = 0.01). Overall, the seroprevalence of T. gondii antibodies in cats in Durango, Mexico, is much lower compared with those reported in other countries.

PMID: 18163360 [PubMed - in process]

Macrophage Migration Inhibitory Factor Is Up-Regulated in Human First-Trimester Placenta Stimulated by Soluble Antigen of Toxo

Am J Pathol. 2007 Dec 28 [Epub ahead of print]

Macrophage Migration Inhibitory Factor Is Up-Regulated in Human First-Trimester Placenta Stimulated by Soluble Antigen of Toxoplasma gondii, Resulting in Increased Monocyte Adhesion on Villous Explants

Vieira Ferro EA, Mineo JR, Ietta F, Bechi N, Romagnoli R, Silva DA, Sorda G, Bevilacqua E, Paulesu LR

From the Instituto de Ciências Biomédicas,* Universidade Federal de Uberlândia, Uberlândia, Brazil; the Dipartimento di Fisiologia, Università degli studi di Siena, Siena, Italy; and the Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAg, interferon-gamma (IFN-gamma), or STAg plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAg (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAg or STAg plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

PMID: 18165264 [PubMed - as supplied by publisher]