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PLoS One ; 10(12): e0144507, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26659253


Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.

Quitinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Quitinases/genética , Quitinases/metabolismo , Cromatografia Líquida , Citocinas/imunologia , Citocinas/metabolismo , Citoplasma/enzimologia , Interações Hospedeiro-Parasita/imunologia , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Cinética , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Espectrometria de Massas em Tandem , Temperatura Ambiente , Toxoplasma/enzimologia , Toxoplasma/fisiologia
PLoS One ; 10(11): e0143087, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26575028


Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Toxoplasmose/prevenção & controle , Vacinação , Animais , Encéfalo/parasitologia , Células Cultivadas , Citocinas/sangue , Escherichia coli , Feminino , Imunidade Celular , Imunidade Humoral , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/biossíntese , Vacinas Protozoárias/biossíntese , Toxoplasmose/imunologia , Toxoplasmose/parasitologia
PLoS One ; 5(5): e10757, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20505765


This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587+/-14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.

Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , Caracteres Sexuais , Animais , Feminino , Proteínas Fúngicas/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/genética , Interleucina-4/metabolismo , Lectinas/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Viabilidade Microbiana , Modelos Imunológicos , Óxido Nítrico/biossíntese , Especificidade de Órgãos/imunologia , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo