Wednesday, October 31, 2007

Modulation of the host cell proteome by Toxo

Infect Immun. 2007 Oct 29; [Epub ahead of print]

Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii

Nelson MM, Jones AR, Carmen JC, Sinai AP, Burchmore R, Wastling JM.

Department of Pre-Clinical Veterinary Science & Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Liverpool, L69 7ZJ. United Kingdom; Division of Infection and Immunity and Sir Henry Welcome Functional Genomics Facility, University of Glasgow, Glasgow, G12 8QQ. United Kingdom; School of Computer Science, University of Manchester, Manchester, M13 9PL. United Kingdom; Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536, USA.

To investigate how intracellular parasites manipulate their host cell environment at the molecular level we have undertaken a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference-gel electrophoresis (DIGE) and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways including glycolysis, lipid and sterol metabolism, mitosis, apoptosis and structural protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, whilst others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one third of all modulated proteins were mitochondrial and this was further investigated by DIGE analysis of a mitochondrial-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested a complex relationship exits between transcription and protein expression that may be partly explained by post-translational modifications of proteins and reveals the importance of investigating protein changes when interpreting transcriptional data. To investigate this further we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion both in terms of protein expression changes and protein modifications and reveal a complex and intimate molecular relationship between host and parasite.

PMID: 17967855 [PubMed - as supplied by publisher]

Tuesday, October 30, 2007

Toxoplasma gondii infection in pregnancy

Braz J Infect Dis. 2007 Oct;11(5):496-506.

Toxoplasma gondii infection in pregnancy

Lopes FM, Gonçalves DD, Mitsuka-Breganó R, Freire RL, Navarro IT

Post-graduate School, Animal Science, Department of Preventive Veterinary Medicine, State University of Londrina.

Toxoplasmosis is caused by an intracellular protozoan, Toxoplasma gondii, which has a wide geographical distribution. The main infection routes are ingestion of cysts from raw or badly-cooked meat, ingestion of oocysts from substrates contaminated with the feces of infected felines and congenital transmission by tachyzoites. The congenital form results in a severe systemic disease, because if the mother is infected for the first time during gestation, she can present a temporary parasitemia that will infect the fetus. Many of the clinical symptoms are seen in congenitally-infected children, from a mild disease to serious signs, such as mental retardation. Early diagnosis during the pregnancy is highly desirable, allowing prompt intervention in cases of infection, through treatment of pregnant women, reducing the probability of fetal infection and consequent substantial damage to the fetus. Conventional tests for establishment of a fetal diagnosis of toxoplasmosis include options from serology to PCR. Prevention of human toxoplasmosis is based on care to avoid infection, understanding the disease and serological exams during gestation. Pregnant women should be tested serologically from three months gestation, until one month after childbirth. Inclusion of serology for congenital toxoplasmosis along with the basic Guthrie test for PKU is of fundamental importance for early diagnosis of infection and so that treatment is initiated, in order to avoid possible sequels in the infant.

PMID: 17962877 [PubMed - in process]

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Int J Parasitol. 2007 Sep 21; [Epub ahead of print]

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Pena HF, Gennari SM, Dubey JP, Su C

Department of Preventive Veterinary Medicine and Animal Health, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, Brazil.

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in São Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits.

PMID: 17963770 [PubMed - as supplied by publisher]

Congenital toxoplasmosis: Priorities for further health promotion action

Public Health. 2007 Oct 25; [Epub ahead of print]

Congenital toxoplasmosis: Priorities for further health promotion action

Elsheikha HM

Division of Veterinary Medicine, The School of Veterinary Medicine and Science, The University of Nottingham, College Road, Sutton Bonington Campus, Leicestershire LE12 5RD, UK.

Toxoplasmosis is a disease of considerable public health impact. As the transmission, occurrence and phenotype of this disease are influenced in a complex way by host genetics, immunity, behaviour and by the agent characteristics, prevention will not be simple. This article aimed to review studies defining seroprevalence of and characteristic sociodemographic, biological and lifestyle risk factors for Toxoplasma gondii infection in pregnant women, to evaluate screening and educational programmes, and to assemble recommendations for combating toxoplasmosis in populations at risk. Electronic databases were searched, using a specific search strategy, from 1975 to 2007. There is a high prevalence of T. gondii antibodies in pregnant women worldwide, with some geographic discrepancies attributed to climatic conditions, local food customs, hygiene, lifestyle and cultural differences. The main risk factors for toxoplasmosis in pregnant women are unsanitary feeding habits, poor immune system, contact with cats, contact with soil, pregnancy, number of births, older age, race, travelling outside the country, drinking beverages prepared with unboiled water, consumption of municipal or uncontrolled (well/spring) water and T. gondii strain virulence. Knowledge of these risk factors helps to identify priorities for further epidemiological work and defines effective preventive measures along five main themes of action: information and health education; screening of pregnant women and infants; limiting harm from risk behaviour; treatment of cases found to be at risk; and vaccination.

PMID: 17964621 [PubMed - as supplied by publisher]

Detection of acute Toxo infection in early pregnancy by IgG avidity and PCR analysis

J Med Microbiol. 2007 Nov;56(Pt 11):1495-9.

Detection of acute Toxoplasma gondii infection in early pregnancy by IgG avidity and PCR analysis

Iqbal J, Khalid N

Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110, Kuwait.

Acute Toxoplasma gondii infection in early pregnancy carries the risk of transmitting the infection to the fetus with serious sequelae. However, serological testing for IgG/IgM anti-Toxoplasma antibodies may fail to differentiate between a recent and past infection. Two hundred and twenty-four Kuwaiti women in their first trimester were screened for IgG/IgM antibodies by the Vitek Immuno Diagnostic Assay System (VIDAS) and VIDAS IgG-avidity tests. On serological screening, 119 (53.1 %) women were positive for IgG antibodies and 31 (13.8 %) for IgM antibodies. Nine of the IgM-positive and 7 IgM-negative women had low-avidity antibodies. However, the IgG-avidity test detected low-avidity antibodies only in 9 (29 %) of the 31 IgM-positive women, suggesting a recent infection; 19 (61.3 %) women had high-avidity antibodies, indicating that the infection was acquired in the distant past. Based on IgM serology alone, at least 31 IgM-positive women may have been wrongly labelled as having acute Toxoplasma infection thus warranting appropriate therapeutic intervention. All the 19 IgM-positive women with high-avidity antibodies were confirmed negative for Toxoplasma DNA on PCR analysis. Compared with PCR analysis, the VIDAS avidity test was a helpful tool for the diagnosis of recent Toxoplasma infection in IgM-negative women with low-avidity antibodies and IgM-positive women with high-avidity antibodies; the specificity was >85 -100 %. It is concluded that the VIDAS avidity test when used in combination with VIDAS IgG/IgM tests is a valuable assay for the exclusion of ongoing or recently acquired T. gondii infection in pregnant women in their first trimester and that it decreases significantly the necessity for follow-up testing and unnecessary therapeutic intervention.

PMID: 17965351 [PubMed - in process]

Friday, October 26, 2007

Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway

Mol Biochem Parasitol. 2007 Sep 16; [Epub ahead of print]

Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway

Braun L, Travier L, Kieffer S, Musset K, Garin J, Mercier C, Cesbron-Delauw MF.
UMR 5163/CNRS-Université Joseph Fourier, Domaine de la Merci, 38700 Grenoble, France.

Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.

PMID: 17959262 [PubMed - as supplied by publisher]

Wednesday, October 24, 2007

Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes

Int J Parasitol. 2007 Sep 15; [Epub ahead of print]

Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes

Iyer LM, Anantharaman V, Wolf MY, Aravind L

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

Comparative genomics of parasitic protists and their free-living relatives are profoundly impacting our understanding of the regulatory systems involved in transcription and chromatin dynamics. While some parts of these systems are highly conserved, other parts are rapidly evolving, thereby providing the molecular basis for the variety in the regulatory adaptations of eukaryotes. The gross number of specific transcription factors and chromatin proteins are positively correlated with proteome size in eukaryotes. However, the individual types of specific transcription factors show an enormous variety across different eukaryotic lineages. The dominant families of specific transcription factors even differ between sister lineages, and have been shaped by gene loss and lineage-specific expansions. Recognition of this principle has helped in identifying the hitherto unknown, major specific transcription factors of several parasites, such as apicomplexans, Entamoeba histolytica, Trichomonas vaginalis, Phytophthora and ciliates. Comparative analysis of predicted chromatin proteins from protists allows reconstruction of the early evolutionary history of histone and DNA modification, nucleosome assembly and chromatin-remodeling systems. Many key catalytic, peptide-binding and DNA-binding domains in these systems ultimately had bacterial precursors, but were put together into distinctive regulatory complexes that are unique to the eukaryotes. In the case of histone methylases, histone demethylases and SWI2/SNF2 ATPases, proliferation of paralogous families followed by acquisition of novel domain architectures, seem to have played a major role in producing a diverse set of enzymes that create and respond to an epigenetic code of modified histones. The diversification of histone acetylases and DNA methylases appears to have proceeded via repeated emergence of new versions, most probably via transfers from bacteria to different eukaryotic lineages, again resulting in lineage-specific diversity in epigenetic signals. Even though the key histone modifications are universal to eukaryotes, domain architectures of proteins binding post-translationally modified-histones vary considerably across eukaryotes. This indicates that the histone code might be "interpreted" differently from model organisms in parasitic protists and their relatives. The complexity of domain architectures of chromatin proteins appears to have increased during eukaryotic evolution. Thus, Trichomonas, Giardia, Naegleria and kinetoplastids have relatively simple domain architectures, whereas apicomplexans and oomycetes have more complex architectures. RNA-dependent post-transcriptional silencing systems, which interact with chromatin-level regulatory systems, show considerable variability across parasitic protists, with complete loss in many apicomplexans and partial loss in Trichomonas vaginalis. This evolutionary synthesis offers a robust scaffold for future investigation of transcription and chromatin structure in parasitic protists.

PMID: 17949725 [PubMed - as supplied by publisher]

Genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers

Exp Parasitol. 2007 Aug 19; [Epub ahead of print]

Toxoplasma gondii: Genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers

Ferreira IM, Vidal JE, Costa-Silva TA, Meira CS, Hiramoto RM, Penalva de Oliveira AC, Pereira-Chioccola VL

Department of Parasitology, Instituto Adolfo Lutz, Av. Dr Arnaldo, 351, 8 andar, CEP 01246-902, São Paulo, SP, Brazil.

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR-amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with recombinant strains (unusual genotype having types I, II and III alleles) 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and recombinant isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as recombinant strains. These data confirm other studies showing the high rate of genetic exchange in T. gondii strains isolated in Brazil.

PMID: 17950282 [PubMed - as supplied by publisher]

Tuesday, October 23, 2007

Effect of melatonin and zinc on the immune response in experimental Toxoplasma retinochoroiditis

Ophthalmologica. 2007;221(6):421-5

Effect of melatonin and zinc on the immune response in experimental Toxoplasma retinochoroiditis

Avunduk AM, Avunduk MC, Baltaci AK, Moğulkoç R

Department of Ophthalmology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey.

OBJECTIVES: To investigate the possible effect of melatonin (MEL) and zinc on the immune response to Toxoplasma gondii retinochoroiditis in the rat model of infection and to establish the possible value of artificial MEL and/or zinc supplementation as adjunctive therapeutic agents in the treatment of T. gondii retinochoroiditis. METHODS: Eighty-four Sprague-Dawley male rats were divided into 12 equal groups. All groups, except controls were infected with T. gondii parasite by intraperitoneal injection. Combinations of zinc-deficient diet, pinealectomy (Px), and artificial zinc and MEL were supplied during a 1-month period. At the end of the experiment, retinal and choroidal total lymphocytes, CD3+, CD4+, and CD8+ cell numbers were counted in histological sections. RESULTS: The highest amount of cellular infiltration (lymphocytes, CD3+, CD4+, CD8+ cells) in the choroid and retina was detected in infected + MEL + zinc-treated rats, and the least amount of cellular infiltration was observed in Px + zinc-deficient diet-treated rats. Although single zinc or MEL supplementation had no significant impact on the cellular infiltration in the retina and choroid in Px rats, combined therapy significantly improved these responses. CONCLUSION: Artificial supplementation of MEL and zinc should be considered as an adjunctive therapy to classic treatment of Toxoplasma retinochoroiditis especially in immunosuppressed and elderly patients if our data are confirmed in a clinical setting. (c) 2007 S. Karger AG, Basel.

PMID: 17947831 [PubMed - in process]

Saturday, October 20, 2007

Kinetic modeling of Toxoplasma gondii invasion

J Theor Biol. 2007 Sep 14; [Epub ahead of print]

Kinetic modeling of Toxoplasma gondii invasion

Kafsack BF, Carruthers VB, Pineda FJ

Department of Microbiology and Immunology, University of Michigan Medical School, 5641 Medical Sciences Bldg. II, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0620, USA; Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, USA; Department of Biostatististics, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205-2179, USA.

The phylum Apicomplexa includes parasites responsible for global scourges such as malaria, cryptosporidiosis, and toxoplasmosis. Parasites in this phylum reproduce inside the cells of their hosts, making invasion of host cells an essential step of their life cycle. Characterizing the stages of host-cell invasion, has traditionally involved tedious microscopic observations of individual parasites over time. As an alternative, we introduce the use of compartment models for interpreting data collected from snapshots of synchronized populations of invading parasites. Parameters of the model are estimated via a maximum negative log-likelihood principle. Estimated parameter values and their 95% confidence intervals (95% CI), are consistent with reported observations of individual parasites. For RH strain parasites, our model yields that: (1) penetration of the host-cell plasma membrane takes 26s (95% CI: 22-30s); (2) parasites that ultimately invade, remain attached three times longer than parasites that eventually detach from the host cells, and (3) 25% (95% CI: 19-33%) of parasites invade while 75% (95% CI: 67-81%) eventually detach from their host cells without progressing to invasion. A key feature of the model is the incorporation of invasion stages that cannot be directly observed. This allows us to characterize the phenomenon of parasite detachment from host cells. The properties of this phenomenon would be difficult to quantify without a mathematical model. We conclude that mathematical modeling provides a powerful new tool for characterizing the stages of host-cell invasion by intracellular parasites.

PMID: 17942124 [PubMed - as supplied by publisher]

Wednesday, October 17, 2007

Congenital toxoplasmosis: late pregnancy infections detected by neonatal screening and maternal serological testing at delivery

Paediatr Perinat Epidemiol. 2007 Nov;21(6):525-31.

Congenital toxoplasmosis: late pregnancy infections detected by neonatal screening and maternal serological testing at delivery

Lago EG, Neto EC, Melamed J, Rucks AP, Presotto C, Coelho JC, Parise C, Vargas PR, Goldbeck AS, Fiori RM

Department of Pediatrics - Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.

The first aim of this study was to determine the prevalence of congenital toxoplasmosis in newborn infants treated by the public health system in Porto Alegre, a city in southern Brazil, using neonatal screening for Toxoplasma gondii-specific IgM. The second aim was to investigate whether the cases detected by this approach could have been identified by the prenatal screening for antibodies to T. gondii that was performed in the same population. A fluorometric assay was used to analyse T. gondii-specific IgM in filter paper specimens obtained from newborn infants for routine screening for metabolic diseases. When the specific IgM was positive, serum samples from the infant and the mother were requested for confirmatory serological testing, and the infant underwent clinical examination. Among 10 000 infants screened for T. gondii-specific IgM, seven filter paper samples were positive, and congenital toxoplasmosis was confirmed in six patients. The prevalence of IgM specific for T. gondii was 6/10 000 [95% CI 2/10 000, 13/10 000]. One infected infant had already been identified in the maternity ward before birth, three had been identified by maternal serology at delivery, and two infants with congenital toxoplasmosis were identified solely through neonatal screening. Although four mothers of the patients with congenital toxoplasmosis received prenatal care, and three mothers had one or two serological tests for T. gondii-specific antibodies (one at first trimester, one at first and second trimesters, and the other at second and third trimesters), they were not identified during pregnancy as infected. Neonatal screening identified cases of infection not detected by obtaining only one or two serum samples from pregnant women for T. gondii serology, mainly when infection was acquired and transmitted in late pregnancy. Maternal serology at delivery and neonatal screening were especially useful in the identification of infants with congenital toxoplasmosis when the mother did not receive regular prenatal serological testing or prenatal care.

PMID: 17937738 [PubMed - in process]

A highly polymorphic family of GPI-anchored surface antigens in Toxo with evidence of developmental regulation

Infect Immun. 2007 Oct 15; [Epub ahead of print]

A highly polymorphic family of GPI-anchored surface antigens in Toxoplasma gondii with evidence of developmental regulation

Pollard AM, Onatolu KN, Hiller L, Haldar K, Knoll LJ

Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706; Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.

The life cycle of the apicomplexan parasite Toxoplasma gondii requires that an infectious cyst develop and be maintained throughout the life of the host. The molecules displayed on the parasite surface are important in controlling the immune response to the parasite. T. gondii has a superfamily of glycosylphosphatidylinositol (GPI)-anchored surface antigens termed the SAG (surface antigen) and SAG-related surface antigens (SRS) that are developmentally regulated during infection. Using a clustering algorithm, we identified a new family of 31 surface proteins that are predicted to be GPI-anchored, but are unrelated to the SAG proteins, thus we named these proteins SAG Unrelated Surface Antigens (SUSA). Analysis of the single nucleotide polymorphism density shows that the members of this family are the most polymorphic genes within the T. gondii genome. Immunofluorescence of SUSA1 and SUSA2, two members of the family, revealed that they are found on the parasite surface. We confirmed that SUSA1 and SUSA2 are GPI anchored by phospholipase cleavage. Analysis of expressed sequence tags (ESTs) revealed that SUSA1 had 22 out of 23 ESTs from chronic infection. Analysis of mRNA and protein confirm that SUSA1 is highly expressed in the chronic form of the parasite. Serum from mice with a chronic T. gondii infection reacts to SUSA1, indicating that SUSA1 interacts with the host immune system during infection. This group of proteins likely represents a new family of polymorphic GPI-anchored surface antigens that are recognized by the host's immune system and whose expression is regulated during infection.

PMID: 17938221 [PubMed - as supplied by publisher]

Tuesday, October 16, 2007

Immunolocalization of an osteopontin-like protein in dense granules of Toxo tachyzoites and its association with the PV

Micron. 2007 Sep 5; [Epub ahead of print]

Immunolocalization of an osteopontin-like protein in dense granules of Toxoplasma gondii tachyzoites and its association with the parasitophorous vacuole

Cortez E, Stumbo AC, Saldanha-Gama R, Villela CG, Barja-Fidalgo C, Rodrigues CA, das Graças Henriques M, Benchimol M, Barbosa HS, Porto LC, Carvalho L

Laboratório Cultura de Células, Departamento de Histologia e Embriologia, IB, Universidade do Estado do Rio de Janeiro, UERJ, Av. Prof. Manoel de Abreu 444, 3° andar, 20550-170, Rio de Janeiro, RJ, Brazil.

Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional glycophosphoprotein, secreted by different cell types, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated in this work by immunofluorescence and immunoelectron microscopy approaches the localization of an OPN-like protein in dense granules of extracellular T. gondii tachyzoites. Western blotting and RT-PCR confirmed this protein expression by the parasites. Our results also showed, after macrophage invasion, an intense positive labeling for OPN-like protein at the sub-apical portion of tachyzoites, the site of dense granules secretion, and the localization of this protein at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we speculate that this protein participates during the parasite interaction process with host cells and parasitophorous vacuole formation.

PMID: 17931871 [PubMed - as supplied by publisher]

Mitochondrial localization of non-histone protein HMGB1 during human endothelial cell-Toxo infection

Cell Biol Int. 2007 Sep 7; [Epub ahead of print]

Mitochondrial localization of non-histone protein HMGB1 during human endothelial cell-Toxoplasma gondii infection

Stumbo AC, Cortez E, Rodrigues CA, Henriques MD, Porto LC, Barbosa HS, Carvalho L

Laboratório Cultura de Células, Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, UERJ, Av. Prof. Manoel de Abreu 444, 3° andar, 20550-170 Rio de Janeiro, RJ, Brazil.

Toxoplasma gondii is an obligate intracellular pathogen, replicating only within a specialized membrane-bounded cytoplasmic vacuole, the parasitophorous vacuole (PV), which interacts with host cell mitochondria. High mobility group box 1 (HMGB1), a known nuclear transcription factor, also may be involved in pathological conditions, whose function is to signal tissue damage. Using confocal microscopy, we have investigated the localization of HMGB1 and the mitochondria performance during interaction between human umbilical vein endothelial cells (HUVEC) and Toxoplasma. Immunofluorescence showed HMGB1 localization in HUVEC tubular mitochondria stained with Mito Tracker (MT). At 2h post-infection, MT labeled spherical structures scattered throughout the cytoplasm and HMGB1 were still present. After 24h of infection, long and tubular structures were localized around PVs and were double labeled by MT and HMGB1, suggesting a structural reorganization of the mitochondria over a long period of infection. For the first time, these results show there is HMGB1 in HUVEC mitochondria and that this protein could be playing a part in mitochondrial DNA events which are important for fission and fusion processes reported here during HUVEC-T. gondii infection.

PMID: 17936030 [PubMed - as supplied by publisher]

Wednesday, October 10, 2007

Drugs designed to inhibit human p38 mitogen-activated protein kinase activation treat Toxo

Antimicrob Agents Chemother. 2007 Oct 8; [Epub ahead of print]

Drugs designed to inhibit human p38 mitogen-activated protein kinase activation treat Toxoplasma gondii and Encephalitozoon cuniculi infection

Wei S, Daniel BJ, Brumlik MJ, Burow ME, Zou W, Khan I, Wadsworth S, Siekierka J, Curiel TJ

Department of Medicine (Hematology and Medical Oncology), Tulane University School of Medicine, 1430 Tulane Avenue SL-78, New Orleans, LA 70112, USA; Department of Surgery, University of Michigan, 1500 East Medical Center Drive, Room TC2101, Ann Arbor, Michigan 48109-0346, USA; San Antonio Cancer Institute, University of Texas Health Sciences Center, 2040 Babcock Rd., suite 201, San Antonio, TX 78229; Louisianna State University Health Sciences Center, New Orleans, LA 70112; Department of Microbiology, Immunology and Tropical Medicine, George Washington University School of Medicine, Washington DC, 20037; The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey.

We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation also inhibited replication of the medically important intracellular parasite, Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful to treat T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with the approved anti-Toxoplasma drugs sulfadiazine or pyrimethamine resulted in improved survival in mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of anti-parasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities of more selective agents.

PMID: 17923491 [PubMed - as supplied by publisher]

Infection of sheep may not prevent foetal infection and abortion in subsequent lambings

Parasitology. 2007 Oct 9;:1-5 [Epub ahead of print

Evidence that primary infection of Charollais sheep with Toxoplasma gondii may not prevent foetal infection and abortion in subsequent lambings

Morley EK, Williams RH, Hughes JM, Thomasson D, Terry RS, Duncanson P, Smith JE, Hide G

Centre for Parasitology and Disease, Biomedical Sciences Research Institute, School of Environment and Life Sciences, University of Salford, Salford M5 4WT, UK.

SUMMARYA study carried out on a sheep farm examined whether Toxoplasma gondii foetal infection and associated abortion occur in successive lambings. We identified 29 ewes that gave birth to lambs on at least 2 successive years over our study period, 2000-2003. Tissue samples from the progeny of these ewes were analysed by PCR to determine infection status with T. gondii. T. gondii-infected lambs were born in 31% of successive pregnancies. T. gondii-positive lambs were aborted in successive pregnancies in 21% of lambings during study period, 2000-2003. The frequency of successive abortions within this flock over the period 1992-2003 was 18%. If a lamb was congenitally infected there was a high risk (69%) that the successive lamb from that ewe would also be congenitally infected. Similarly, if a lamb was aborted there was a high risk (55%) of abortion in the next lamb produced. These data suggest that life-long immunity to T. gondii infections may not always be acquired following an initial infection and raises the question as to whether the mechanisms of T. gondii transmission prior to and during ovine pregnancies are fully understood.

PMID: 17922930 [PubMed - as supplied by publisher]

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma activity induced by lipopolysaccharides

Int J Parasitol. 2007 Sep 6; [Epub ahead of print]

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Authier H, Cassaing S, Bans V, Batigne P, Bessières MH, Pipy B

Laboratoire des macrophages, Médiateurs de l’Inflammation et Interactions Cellulaires, Université Paul Sabatier Toulouse III, EA2405, INSERM IFR31 BP84225, 31432 Toulouse Cedex 4, France.

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.

PMID: 17923133 [PubMed - as supplied by publisher]

TAP-1 indirectly regulates CD4+ T cell priming in Toxo infection

J Exp Med. 2007 Oct 8; [Epub ahead of print]

TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-{gamma} production

Goldszmid RS, Bafica A, Jankovic D, Feng CG, Caspar P, Winkler-Pickett R, Trinchieri G, Sher A

Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892.

To investigate if transporter associated with antigen processing (TAP)-1 is required for CD8(+) T cell-mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1(-/-), CD8(-/-), and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1(-/-) mice displayed greater susceptibility than CD8(-/-), beta(2)-microglobulin(-/-) (beta(2)m(-/-)), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1(-/-) mice correlated with a reduction in the frequency of activated (CD62L(low) CD44(hi)) and interferon (IFN)-gamma-producing CD4(+) T cells. Interestingly, infected TAP-1(-/-) mice also showed reduced numbers of IFN-gamma-producing natural killer (NK) cells relative to WT, CD8(-/-), or beta(2)m(-/-) mice, and after NK cell depletion both CD8(-/-) and WT mice succumbed to infection with the same kinetics as TAP-1(-/-) animals and displayed impaired CD4(+) T cell IFN-gamma responses. Moreover, adoptive transfer of NK cells obtained from IFN-gamma(+/+), but not IFN-gamma(-/-), animals restored the CD4(+) T cell response of infected TAP-1(-/-) mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-gamma-producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.

PMID: 17923502 [PubMed - as supplied by publisher]

Tuesday, October 09, 2007

Chemical inactivation of Toxoplasma oocysts in water

J Parasitol. 2007 Aug;93(4):925-31.

Chemical inactivation of Toxoplasma gondii oocysts in water

Wainwright KE, Miller MA, Barr BC, Gardner IA, Melli AC, Essert T, Packham AE, Truong T, Lagunas-Solar M, Conrad PA

Department of Pathology, Microbiology and Immunology, 1 Shields Avenue, School of Veterinary Medicine, University of California, Davis, California 95616, USA.

The protozoan parasite Toxoplasma gondii is increasingly recognized as a waterborne pathogen. Infection can be acquired by drinking contaminated water and conventional water treatments may not effectively inactivate tough, environmentally resistant oocysts. The present study was performed to assess the efficacy of 2 commonly used chemicals, sodium hypochlorite and ozone, to inactivate T. gondii oocysts in water. Oocysts were exposed to 100 mg/L of chlorine for 30 min, or for 2, 4, 8, 16, and 24 hr, or to 6 mg/L of ozone for 1, 2, 4, 8, or 12 min. Oocyst viability was determined by mouse bioassay. Serology, immunohistochemistry, and in vitro parasite isolation were used to evaluate mice for infection. Initially, mouse bioassay experiments were conducted to compare the analytical sensitivity of these 3 detection methods prior to completing the chemical inactivation experiments. Toxoplasma gondii infection was confirmed by at least 1 of the 3 detection methods in mice inoculated with all doses (10(5)-10(0)) of oocysts. Results of the chemical exposure experiments indicate that neither sodium hypochlorite nor ozone effectively inactivate T. gondii oocysts, even when used at high concentrations.

PMID: 17918377 [PubMed - in process]

Longer pregnancy and slower fetal development in women with latent "asymptomatic" toxoplasmosis

BMC Infect Dis. 2007 Oct 4;7(1):114 [Epub ahead of print]

Longer pregnancy and slower fetal development in women with latent "asymptomatic" toxoplasmosis

Kankova S, Flegr J

ABSTRACT: BACKGROUND: The purpose of this study was to confirm that women with latent toxoplasmosis have developmentally younger fetuses at estimated pregnancy week 16 and to test four exclusive hypotheses that could explain the observed data. METHODS: In the present retrospective cohort study we analysed by the GLM (general linear model) method data from 730 Toxoplasma-free and 185 Toxoplasma-infected pregnant women. RESULTS: At pregnancy week 16 estimated from the date of the last menstruation, the mothers with latent toxoplasmosis had developmentally younger fetuses based on ultrasound scan (P = 0.014). Pregnancy of Toxoplasma-positive compared to Toxoplasma-negative women was by about 1.3 days longer, as estimated both from the date of the last menstruation (P = 0.015) and by ultrasonography (P = 0.025). CONCLUSION: The most parsimonious explanation for the observed data is retarded fetal growth during the first weeks of pregnancy in Toxoplasma-positive women. The phenomenon was only detectable in multiparous women, suggesting that the immune system may play some role in it.

PMID: 17916246 [PubMed - as supplied by publisher]

Friday, October 05, 2007

Toxofilin forms a ternary complex with an antiparallel actin dimer

Proc Natl Acad Sci U S A. 2007 Oct 2; [Epub ahead of print]

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Lee SH, Hayes DB, Rebowski G, Tardieux I, Dominguez R

Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085; Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472.

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69-196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.

PMID: 17911258 [PubMed - as supplied by publisher]

Wednesday, October 03, 2007

Phosphoinositide 3-Kinase Dependent, MyD88-Independent Induction of CC-Type Chemokines Characterizes the Macrophage Response to High Virulence Toxo

Infect Immun. 2007 Oct 1; [Epub ahead of print]

Phosphoinositide 3-Kinase Dependent, MyD88-Independent Induction of CC-Type Chemokines Characterizes the Macrophage Response to High Virulence Toxoplasma gondii

Lee CW, Sukhumavasi W, Denkers EY

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

Chemokines play an important role in inflammation and infection due to their ability to recruit cells of innate and adaptive immunity. Here, we examined mouse macrophage chemokine responses during intracellular infections with high and low virulence Toxoplasma gondii strains. The high virulence type I strain RH induced a large panel of CC-type chemokines, whereas responses elicited by PTG (type II) and M7741 (type III) were much weaker. Strikingly, the T. gondii-induced chemokine response occurred independently of signaling through Toll-like receptor (TLR) adaptor MyD88. Instead, production of chemokines during infection was heavily dependent upon phosphoinositide (PI) 3-kinase signaling pathways. Because infection with type I strains such as RH results in an uncontrolled proinflammatory cytokine response, we hypothesize that this virulence phenotype is a consequence of early strong induction of chemokines by type I, but not type II or III, Toxoplasma strains.

PMID: 17908814 [PubMed - as supplied by publisher]

Tuesday, October 02, 2007

Virulence of tachyzoites in serum free media at different temperatures

Exp Parasitol. 2007 Jul 13; [Epub ahead of print]

Toxoplasma gondii: Virulence of tachyzoites in serum free media at different temperatures

Diab MR, El-Bahy MM

Organization for Vaccines and Biological Products (VACSERA), El-Agoza, Giza, Egypt.

Highly virulent Toxoplasma gondii tachyzoite multiplication was recorded on the 4th and 5th days post cultivation (dpc) in seven selected cell lines either with or without fetal calf serum (FCS) in the maintenance media. The multiplication rate was slightly lower in the absence of FCS. The cell line mono-layers collapsed dying by the 6th day of infection both in presence or absence of FCS at 37 degrees C. Carcinoma of human larynx (Hep2) and Madian Darby Bovine Kidney (MDBK) cell lines were the most suitable for in vitro multiplication, followed by that of African green monkey kidney cells (VERO), pooled kidney from 1-day-old hamster (BHK), rabbit kidney cells (RK13) and human rhabdomyosarcoma (RDA), while Chicken embryo cells (CER) were the least suitable. In absence of FCS, CER, BHK, Hep2, RDA and MDBK were able to maintain virulent tachyzoites at +4 degrees C for 14 days. The infectivity of the tachyzoites was however lower, killing 40% of the inoculated mice. Tachyzoites survived at room temperature, in the dark, for 14 days in Hep2, RDA and MDBK. However, Hep2 was the only one able to keep virulent tachyzoites until 21 dpc at room temperature and at +4 degrees C. Hep2 propagated tachyzoites were still alive but with low infectivity up to 28 dpc. The cell-lines failed to support the development of tachyzoites after 7 dpc at 37 degrees C and after the 35 dpc at lower temperatures.

PMID: 17904554 [PubMed - as supplied by publisher]

Synthesis, anti-Toxo and antimicrobial activities of...

Bioorg Med Chem. 2007 Sep 18; [Epub ahead of print]

Synthesis, anti-Toxoplasma gondii and antimicrobial activities of benzaldehyde 4-phenyl-3-thiosemicarbazones and 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids

de Aquino TM, Liesen AP, da Silva RE, Lima VT, Carvalho CS, de Faria AR, de Araújo JM, de Lima JG, Alves AJ, de Melo EJ, Góes AJ

Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife 50670-901, Brazil.

In the present communication, a new series of 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids (2a-p) have been synthesized. Benzaldehyde 4-phenyl-3-thiosemicarbazones substituted (1a-p) were also obtained and used as intermediate to give the title compounds. All synthesized compounds were characterized by IR, (1)H and (13)C NMR. The in vitro anti-Toxoplasma gondii activity of 1a-p and 2a-p was evaluated. The 4-thiazolidinones (2a-p) were screened for their in vitro antimicrobial activity. For anti-Toxoplasma gondii activity, in general, all compounds promoted decreases in the percentage of infected cells leading to parasite elimination. These effects on intracellular parasites also caused a decrease in the mean number of tachyzoites. In addition, most of the 4-thiazolidinones showed more effective toxicity against intracellular parasites, with IC(50) values ranging from 0.05 to 1mM. According to results of antimicrobial activity, compounds 2f, 2l, and 2p showed best activity against Mycobacterium luteus, 2c was more active against Mycobacterium tuberculosis, and 2g, 2l, and 2n showed same activity as nistatin (standard drug) against Candida sp. (4249).

PMID: 17905587 [PubMed - as supplied by publisher]

A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice

Microbes Infect. 2007 Jul 15; [Epub ahead of print]

A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice

Beauvillain C, Ruiz S, Guiton R, Bout D, Dimier-Poisson I

Université François-Rabelais, INRA, UMR 0483 Université-INRA d'Immunologie Parasitaire et Vaccinologie, IFR des Agents Transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31, Avenue Monge, 37200 Tours, France; Unité Mixte Institut National de la Santé et de la Recherche Médicale 564, University Hospital, 4, rue Larrey, 49933 Angers, France; Immunology and Allergology Laboratory, University Hospital, Angers, France.

Our results show that exosomes secreted by SRDC pulsed in vitro with Toxoplasma gondii-derived antigens (Exo-TAg) induced protective responses against infection with the parasite in both syngeneic and allogeneic mice. After oral infection, syngeneic CBA/J mice exhibited significantly fewer cysts in their brains and allogeneic C57BL/6 mice survived. This protection was associated with strong humoral responses in vivo in serum from both CBA/J and C57BL/6 mice, and with high levels of anti-TAg IgA antibodies in intestinal secretions from CBA/J mice alone. Furthermore, strong cellular responses in vivo were observed in both mouse models. Cellular proliferation was associated with cytokines production by spleen and mesenteric lymph node cells. The results presented here show that exosomes are nucleic acid free vesicles that are able to induce immune responses correlated with protection against parasitic infections in both syngeneic and allogeneic mice. They could constitute an efficient tool for use in vaccination and antitumor strategies based on exosomes.

PMID: 17905628 [PubMed - as supplied by publisher]