Systems-based analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle.

UNLABELLED: 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.

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.

Síntesis de renina por células renales durante la inhibición crónica de la enzima convertidora de la angiotensina I / Renin synthesis by renal cells during chronic inhibition of angiotensin I converting enzyme

Se realizó un estudio sobre la síntesis de renina y las modificaciones en el número de células que se produce durante la inhibición crónica de la enzima convertidora de la Ang II en ratones. Veinte ratones CF1, hembras de 15 días de edad, inmediatamente después del destete, recibieron 20 mg/l de maleato de enalapril (ME) con el agua de beber durante 18 meses y se compararon con un grupo control. El tejido renal fue procesado y estudiado con técnicas inmunohistoquímicas con microscopía óptica y electrónica utilizando un anticuerpo policlonal antirrenina y se realizó hibridización in situ para rasteo de ARNm de renina con una sonda marcada con diagoxigenina. Los parámetros calculados fueron el número de aparatos yuxtaglomerulares (MAYG), de arteriolas aferentes (AA) y vasos arcuatos (VA) inmunomarcados con antirrenina y con antidigoxigenina. Con microscopía electrónica se determinó el número de partículas de oro por gránulo de renina. Se demostró aumento del número de células productoras de renina en los animales que recibieron crónicamente ME, más allá del AYG y de la AA ya que se observó marcación en vasos arcuatos. La media de porcentagem MAYG y porcentagemMAA fue menor en los animales controles (Con) que en los tratados (TRA). No se marcaron los VA en el grupo Con, y sí en los animales Tra. La distribución del ARN fue diferente en los animales con inhibición del SRA que en los controles. Los signos de hibridización fueron significativamente menores en los animales Con, tanto el porcentagemSAYG, el porcentagemSAA. En los VA sólo se observaron signos en los animales tratados porcentagem SVA. La cantidad de partículas de oro en las células productoras de renina fue diferente entre los dos grupos de animales. La media del número de partículas de oro en los gránulos de renina perteneciente al AYG de los animales Con. fue significativamente menor que en los tratados. En este trabajo se demostró que los animales que tienen inhibido crónicamente la producción de Ang II por ME, aumentan la síntesis, el número y la localización de células productoras de renina en la vasculatura renal, recapitulando situaciones que ocurren durante el desarrollo embriológico de mamíferos y en vertebrados primitivos adultos

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