Targeted Disruption of Toxoplasma gondii Serine Protease Inhibitor 1 Increases Bradyzoite Cyst Formation In Vitro and Parasite Tissue Burden in Mice

As an intracellular protozoan parasite, Toxoplasma gondii is likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities. T. gondii serine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces two TgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigate TgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles with Dolichos biflorus lectin under conditions promoting in vitro differentiation. The differentiation phenotype can be partially complemented by either TgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and this in vivo phenotype is also complemented by either TgPI1 isoform. These results demonstrate that TgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases

Systems-Based Analysis of the \u3cem\u3eSarcocystis neurona\u3c/em\u3e Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies

Toxoplasma Gondii : producción de anticuerpos monoclonales y su utilización en el estudio del rol biológico de antígenos del parásito

Fil:Pszenny, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A simple and economic slide micro-immunoenzymatic (Micro-SIA) test for epidemiological studies of toxoplasmosis

A slide micro-immunoenzymatic assay (micro-SIA) to detectantibodies to non-particulate Toxoplasma gondii antigens is described. This assay allows the diagnosis of toxoplasmosis infection in about 1 hr. Twenty-four determinations can be performed per slide. Five hundred ng of antigen and 5 or 10 µl drop of each reactive are necessary per well. The clear contrast of colours obtained for negative and positive sera after the test is finished, allows direct discrimination of the results. However, it is possible to quantify the results of the reaction using a minireader. Sera dilution cutoff value, determined as themost frequent titre for the general population, is 1:100. The toxoplasma micro-SIA correlates well with indirect immunofluorescence (IIF), its sensitivity is atleast three times as much as IIF. The test has an intra and inter assay variation coefficient of 5.46 per cent and of 6.24 per cent respectively. Sera obtained at random from argentinian people were analyzed and a 56 per cent of infection was found. The main features of the Toxoplasma micro-SIA are its simplicity, sensitivity, reproducibility, and the virtual absence of background making it very suitable for screening tests

High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis

Fil: Nigro, Mónica. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Kaufer, Federico. Hospital Alemán. Laboratorio de Toxoplasmosis; Argentina.Fil: Carral, Liliana. Hospital Alemán. Laboratorio de Toxoplasmosis; Argentina.Fil: Angel, Sergio O. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Pszenny, Viviana. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2(196-561) fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2(196-561) was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2(196-561) became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2(196-561) keeps its diagnostic value in contrast with the insoluble protein

Characterisation of a novel interspersed Toxoplasma gondii DNA repeat with potential uses for PCR diagnosis and PCR-RFLP analysis

Fil: Echeverria, Pablo C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Rojas, Paola A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.Fil: Ángel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología. Departamento de Parasitología Sanitaria; Argentina.A novel Toxoplasma gondii interspersed repeat element (TgIRE), present in most of the tachyzoite chromosomes, was characterised. Two regions on the TgIRE sequence showed high identity to two different T. gondii expressed sequence tag cDNAs of unknown function, which seems to be TgIRE pseudogenes. Two set of primers were designed, 2-2' and 2-3, that amplify products of 1.02 and 0.62 kb, respectively. T. gondii DNA from RH and Me49 strains was amplified with TgIRE 2-2' primers, and the respective 1.02 kb products were digested with several endonucleases. Different fragment patterns by gel electrophoresis were found only with MboI. Sensitivity analysis revealed that the set 2-3 was more sensitive than 2-2', detecting by gel visualisation the amount of DNA equivalent to 1 and 10 parasites, respectively

Expression of a cDNA encoding a Toxoplasma gondii protein belonging to the heat-shock 90 family and analysis of its antigenicity

A cDNA clone (Tgzy85d11.r1) obtained from the Toxoplasma Expressed Sequence Tag project was chosen due to its homology with proteins of the heat shock 90 family. The cDNA encodes 137 amino acids of the C-terminal portion of the Toxoplasma Hsp90 protein (TgHsp90). Serum samples obtained from orally infected BALB/c and C57BL/6 mice showed reactivity against a recombinant TgHsp90 (rTgHsp90) after 8 weeks postinfection. Isotype analysis showed an anti-rTgHsp90 IgG2a/IgG3 response in infected BALB/c and antirTgHsp90 IgG1/IgG2a/IgG2b response in infected C57BL/6 mice. Serum samples from individuals chronically and putative acutely infected with T. gondii showed a similar anti-rTgHsp90 IgG response. Our work identifies TgHsp90 as a novel parasite antigen that seems to elicit a higher relation of anti-TgHsp90/anti-T. gondii IgGs during chronic infection in comparison with the acute stage

Subcellular localization and post-secretory targeting of TgPI, a serine proteinase inhibitor from Toxoplasma gondii

Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Ledesma, Bibiana A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Matrajt, Mariana. Department of Biology, University of Pennsylvania, Philadelphia; Estados Unidos.Fil: Duschak, Vilma G. Instituto de Investigaciones Biotecnológicas, UNSAM, Provincia de Buenos Aires; Argentina.Fil: Bontempi, Esteban J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Dubremetz, Jean-François. UMR5539 CNRS, Université de Montpellier II, Montpellier; Francia.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization

Fil: Bontempi, Esteban. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Garcia, G. A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Buschiazzo, A. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de Gral. San Martín, INTI; Argentina.Fil: Henriksson, J. Department of Medical Genetics and Pathology, Uppsala University, Suecia.Fil: Pravia, Carlos. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Ruiz, Andrés Mariano. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Pettersson, Ulf. Department of Medical Genetics and Pathology, Uppsala University; Suecia.Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates