Envolvimento de mitocôndrias e do retículo endoplasmático da célula muscular esquelética na cistogênese de Toxoplasma gondii / Involvement of mitochondria and endoplasmic reticulum in skeletal muscle cell on Toxoplasma gondii cystogenesis

T. gondii é parasito intracelular obrigatório, agente etiológico datoxoplasmose, doença com ampla distribuição mundial. Os transtornos mais severos(fase aguda) acometem pacientes imunocomprometidos. Hospedeirosimunologicamente sadios, uma vez infectados, apresentam cistos teciduais (fasecrônica) de modo perene. Estudos sugerem que por possuírem um eficientemetabolismo energético, os principais tecidos eleitos para a cistogênese do T.gondii, são o nervoso e o muscular esquelético. O presente trabalho dedicou-se aoestudo das associações de mitocôndrias e do retículo endoplasmßtico (RE) àmembrana do vacúolo parasitóforo (MVP) e à parede cística. Para tanto, foramutilizados bradizoítos e taquizoítos da cepa ME49 (tipo II) e culturas primßrias decélula muscular esquelética (CME) e da linhagem C2C12. Nossas estratégiasmetodológicas contemplaram microscopia de fluorescência, microscopia eletrônicade transmissão, respirometria de alta resolução e ensaios de efeito de um inibidor dafosforilação oxidativa (ISA-34) sobre a cistogênese. Os dados obtidos apontam aocorrência de: (i) associações entre mitocôndrias com a parede cística; (ii) aspectospeculiares ultraestruturais decorrentes de associações entre mitocôndrias e RE(rugoso e liso) da CME com a MVP de vacúolos contendo bradizoítos; (iii)manutenção do metabolismo mitocondrial da CME pelo T. gondii, durante a fasecrônica; (iv) efeito inibitório do composto ISA-34 sobre o desenvolvimento de cistosteciduais. Estes resultados, além de iniciarem uma linha de pesquisa inédita arespeito das respostas do metabolismo energético da CME frente à cistogênese deT. gondii, também abrem novas perspectivas para uma terapia alternativa voltadapara a fase crônica da toxoplasmose...

Toxoplasma gondii is an obligatory intracellular parasite, agent oftoxoplasmosis, disease with a worldwide distribution. The most severe disorders(acute phase) affect immunocompromised patients. Immunologically healthyindividuals, once infected, develop tissue cysts (chronic phase) that can persist forthe host life span. Studies suggest that an efficient energetic metabolism, as innervous and skeletal muscle tissues, leads to the development of T. gondiicystogenesis. The present work aims the study of the association of skeletal musclecell (SkMC) mitochondria and endoplasmic reticulum (ER) to the parasitophorousvacuole membrane (PVM) and to the cyst wall (CW). Bradyzoites and tachyzoitesfrom ME49 strain (type II) of T. gondii and SkMC cultures and C2C12 cell line wereused. The methodological strategies employed were fluorescence microscopy,transmission electron microscopy, high-resolution respirometry and assay using ISA-34, an inhibitor of oxidative phosphorylation. Our data point out: (i) associationsbetween mitochondria and CW; (ii) ultrastructural aspects of the association of SkMCmitochondria and ER (rough and smooth) with PVM of bradyzoite-containingvacuoles; (iii) maintenance of SkMC mitochondrial metabolism by T. gondii and, (iv)inhibitory effect of ISA-34 on the tissue cysts development. These results stimulatefurther investigation concerning the response of SkMC energy metabolism duringcystogenesis of T. gondii and also open novel perspectives for an alternative therapyagainst toxoplasmosis chronic phase...

Efeitos in vitro e de 3-bromopiruvato e atovaquona na infecção por Toxoplasma gondii em células LLC-MK2 / Effect in vitro of 3-bromopyruvate and atovaquone on infection of toxoplasma gondii in llc-mk2 cells

Toxoplasma gondii causa uma infecção comumente assintomática, porémpode se apresentar de forma grave durante a gravidez e em pacientesimunocomprometidos. A terapia atual para a toxoplasmose é restrita contrataquizoítos, e possuem pouco ou nenhum efeito sobre bradizoítos, que são mantidosem cistos teciduais como fonte recrudescente da infecção. Com isso, novasalternativas terapêuticas vêm sendo propostas, como o uso da Atovaquona, queapresentou alguma eficácia sobre taquizoítos e bradizoítos em cistos teciduais.Neste trabalho, foi estudado o efeito de 3-BrPA, um composto utilizado em testessobre células cancerígenas, durante a interação de células LLC-MK2 infectadas comtaquizoítos da cepa RH de T. gondii. Quanto à célula hospedeira não se observouefeito do composto sobre a proliferação e viabilidade celular. A avaliação dainterferência de 3-BrPA sobre o crescimento in vitro do T. gondii evidenciou umaredução na proliferação intracelular do parasito de cerca de 55 por cento após 24 h detratamento e 61 por cento após 48 h. O desenvolvimento intracelular do parasito, analisadopor MEV, apresentou características morfológicas comumente encontradas emcistos teciduais. A incubação das culturas com lectina DBA confirmou odesenvolvimento de cistos e por MET se evidenciou a presença de bradizoítos. Alémdisso, foram revelados parasitos degradados e a influência do composto sobre aendodiogenia. Outra abordagem adotada foi o tratamento de culturas infectadas coma combinação de 3-BrPA e Atovaquona, que resultou em uma redução de parasitosintracelulares de 73 por cento após 24 h de tratamento e 71 por cento após 48 h, em comparação aocontrole, além da ausência da formação de parede cística nessas culturas...

Toxoplasma gondii usually causes an asymptomatic infection, but it can present severityduring pregnancy and in immunocompromised patients. Current therapies fortoxoplasmosis are restricted only against tachyzoites, and have little or no effect onbradyzoites, which are kept in tissue cysts like source of the infection recrudescent.Consequently, new therapeutic alternatives have been proposed, as the use ofAtovaquone that showed some efficacy against tachyzoites and bradyzoites in tissuecysts. In this work, we propose to study the effect of 3-BrPA, a compound that is beingtested against cancer cells, on the infection of LLC-MK2 cells with tachyzoites of T.gondii (RH strain). No effect of 3-BrPAon host cell proliferation and viability wasobserved. Evaluation of 3-BrPA interference on in vitro growth of T. gondii showed areduction in intracellular parasite proliferation about 55 por cento after 24 h of treatment, and61 por cento after 48 h. Intracellular development of parasite, analyzed by SEM, showedmorphological characteristics commonly found in tissue cysts. Incubation of cultureswith DBA lectin confirmed the development of cysts and TEM showed the presence ofbradyzoites. Moreover, we revealed the presence of degraded parasites and theinfluence of compound on endodyogeny. Another approach used was the treatment ofinfected cultures with combination of 3-BrPA and Atovaquone. This resulted in thereduction of intracellular parasites of 73 por cento after 24 h of treatment and 71 percent after 48 h,compared to control, besides the absence of cyst wall formation in these cultures...

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.

Toxoplasmosis sistémica en un canguro rojo (Macropus rufus) cautivo / Systemic toxoplamosis in a captive red kangaroo (Macropus rufus)

Los objetivos del estudio fueron presentar y documentar los hallazgos histopatológicos de toxoplasmosis sistémica en un canguro rojo (Macropus rufus) mantenido en cautiverio donde se describen los hallazgos macro y microscópicos encontrados y los análisis adicionales realizados. En el laboratorio de histopatología animal (Universidad de los Llanos) se recibieron muestras de tejidos fijados en formol tamponado, al 10% que procedían de un ejemplar macho de Macropus rufus, de ocho años de edad y 50 kg de peso corporal. Las muestras se procesaron mediante métodos rutinarios para microscopía óptica. Los cortes histológicos de 3-4 mm de grosor se colorearon con Hematoxilina-Eosina (H&E) y se realizó en algunos cortes la tinción de Ácido Periódico Schiff (PAS), PCR e IHQ. Al análisis histopatológico se encontró una toxoplasmosis sistémica asociada a quistes de protozoarios con inmunoreactividad positiva para T. gondii. La detección de T gondii en tejidos en formalina fue hecha usando dos ensayos de PCR que señalaban segmentos de ADN de diferentes secuencias repetitivas encontradas en T gondii y la IHQ confirmo lo hallado por PCR. Histopatológicamente se diagnosticó infección crónica por protozoarios eucoccideos de la familia Sarcocystidae. El diagnóstico etiológico fue de toxoplasmosis.

The objetives of this study were to present and document the hystopathologycal findings of systemic toxoplamosis in a captive red kangaroo (Macropus rufus) which described macro and microscopic findings of the hystopathological analysis. In the laboratory of animal histopathology (Universidad de los Llanos) formalin fixed tissue specimens were received, from a captive male Macropus rufus, who was eight years old and weighed 50 kg. The samples were processed by usual methods for optical microscopy. The histological sections of 3-4 mm thick were colored with Hematoxilin-Eosin (H&E) and then some samples stained with Periodic Acid Schiff (PAS), and processed by PCR and IHQ. Once the histopathological analysis was performed systemic toxoplasmosis was associated to protozoa cysts immunoreactives to T. gondii. The molecular detection of T. gondii in formalin fixed tissues was made using two PCR tests and confirmated by IHQ. Histopathologically a chronic infection by an eucoccideo protozoa from the Sarcocystidae family was diagnosed. The etiologic diagnosis was toxoplasmosis.

Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review

Parasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immunocompromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research.

Toxoplasmose ocular: utilizaçäo de reaçäo imuno-histoquimica para diagnóstico / Ocular toxoplasmosis: utilization of immunohistochemistry in the diagnosis of ocular toxoplasmosis

O diagnóstico de toxoplasmose ocular baseia-se geralmente em dados clinicos e laboratoriais bastante conhecidos. Porém, casos com apresentaçäo clinica atipica ocorrem, principalmente em pacientes imunossuprimidos, trazendo entäo ao médico clinico dificuldades diagnósticas. Os autores descrevem o caso de uma paciente do sexo feminino, com uma doenca ocular bilateral, de tres anos de duracao, em evoluçäo e sem diagnóstico etiológico estabelecido. Após exaustiva investigaçäo clinica e laboratorial e utilizaçäo de vários medicamentos, sem sucesso clinico, optou-se pela enucleaçäo de olho esquerdo, ja atrofico...