10 research outputs found
Toxoplasma gondii chitinase induces macrophage activation
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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 degrees 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.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 pathoge1012112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/10741-8]2013/10741-8SEM INFORMAÇÃOThis study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2013/10741-8). Additional financial help was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio ao Ensino, Pe
Production and composition evaluation of extracellular vesicles from Aspergillus fumigatus
Aspergillus fumigatus é considerado como o principal fungo causador de infecções em indivíduos imunocomprometidos, tendo grande importância médica. O sucesso das infecções depende da secreção de variadas moléculas ao espaço extracelular, mediadas, por exemplo, por meio de vesículas extracelulares (VEs). VEs fúngicas contém proteínas, lipídios, pigmentos, polissacarídeos, DNA, RNA e outras moléculas que podem ser cruciais na fisiopatologia fúngica, mas existem poucos estudos que demonstrem a composição de VEs por fungos do gênero Aspergillus. Desta forma, nosso objetivo foi avaliar a produção e composição de VEs produzidas por A. fumigatus em meio líquido e sólido. Nossos resultados confirmam que A. fumigatus produz VEs em ambos os meios, com maior rendimento em meio sólido, e demonstram presença de metabólitos secundários, diversas populações de RNAs e proteínas. Entre as moléculas encontradas, pudemos identificar moléculas relacionadas à virulência fúngica, contra insetos, protozoários e células de mamíferos, à modulação da resposta imune de hospedeiros e, consequentemente, à sobrevivência do fungo. As descobertas relatadas neste estudo poderão ajudar a elucidar a função das VEs produzidas por A. fumigatus e entender os processos biológicos aos quais elas estão relacionadas.Aspergillus fumigatus is a fungus considered to be the main causative agent of infections in immunocompromised individuals, with great medical importance. The success of these infections depends on the secretion of several molecules to the extracellular space, mediated, for example, by extracellular vesicles (EVs). Fungal EVs contain proteins, lipids, pigments, polysaccharides, DNA, RNA and other molecules that can be crucial to fungal pathophysiology, however there are few studies demonstrating the composition of EVs produced by the Aspergillus genus. Thus, our objective was to evaluate the production and composition of EVs produced by A. fumigatus in liquid and solid media. Our results confirm that A. fumigatus produces EVs in both media, with higher yield in solid media, and demonstrate the presence of secondary metabolites, different populations of RNAs and proteins. Among the molecules found, we were able to identify molecules related to fungal virulence, with action against insects, protozoa and mammalian cells, to the modulation of host\'s immune response and, consequently, to the fungus survival. The findings reported in this study may help to elucidate the function of EVs produced by A. fumigatus and to understand the biological processes to which they are related
Influence of N-glycans on Expression of Cell Wall Remodeling Related Genes in Paracoccidioides brasiliensis Yeast Cells
Toxoplasma gondii Chitinase Induces Macrophage Activation.
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
Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects
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Previous issue date: 2017-12-06University of Sao Paulo. Ribeirao Preto Medical School. Department of Biochemistry and Immunology. Ribeirao Preto, SP, Brasil.Yeshiva University. Albert Einstein College of Medicine. Department of Microbiology and Immunology. New York, NY, EUA.University of Sao Paulo. Ribeirao Preto Medical School. Department of Cellular and Molecular Biology. Ribeirao Preto, SP, Brasil.Yeshiva University. Albert Einstein College of Medicine. Department of Microbiology and Immunology. New York, NY, EUA.University of Sao Paulo. Ribeirao Preto Medical School. Department of Biochemistry and Immunology. Ribeirao Preto, SP, Brasil.University of Sao Paulo. Ribeirao Preto Medical School. Department of Biochemistry and Immunology. Ribeirao Preto, SP, Brasil.University of Sao Paulo. Ribeirao Preto Medical School. Department of Cellular and Molecular Biology. Ribeirao Preto, SP, Brasil.University of Sao Paulo. Ribeirao Preto Medical School. Department of Cellular and Molecular Biology. Ribeirao Preto, SP, Brasil.University of Sao Paulo. Ribeirao Preto Medical School . Department of Internal Medicine. Ribeirao Preto, SP, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes. Rio de Janeiro, RJ, Brasil.University of Sao Paulo. Ribeirao Preto Medical School. Department of Cellular and Molecular Biology. Ribeirao Preto, SP, Brasil.Johns Hopkins Bloomberg School of Public Health. Department of Molecular Microbiology and Immunology. Baltimore, MD, EUA.Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian β-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host
New Insights on Dietary Polyphenols for the Management of Oxidative Stress and Neuroinflammation in Diabetic Retinopathy
Diabetic retinopathy (DR) is a neurodegenerative and vascular pathology that is considered one of the leading causes of blindness worldwide, resulting from complications of advanced diabetes mellitus (DM). Current therapies consist of protocols aiming to alleviate the existing clinical signs associated with microvascular alterations limited to the advanced disease stages. In response to the low resolution and limitations of the DR treatment, there is an urgent need to develop more effective alternative therapies to optimize glycemic, vascular, and neuronal parameters, including the reduction in the cellular damage promoted by inflammation and oxidative stress. Recent evidence has shown that dietary polyphenols reduce oxidative and inflammatory parameters of various diseases by modulating multiple cell signaling pathways and gene expression, contributing to the improvement of several chronic diseases, including metabolic and neurodegenerative diseases. However, despite the growing evidence for the bioactivities of phenolic compounds, there is still a lack of data, especially from human studies, on the therapeutic potential of these substances. This review aims to comprehensively describe and clarify the effects of dietary phenolic compounds on the pathophysiological mechanisms involved in DR, especially those of oxidative and inflammatory nature, through evidence from experimental studies. Finally, the review highlights the potential of dietary phenolic compounds as a prophylactic and therapeutic strategy and the need for further clinical studies approaching the efficacy of these substances in DR management
Amino acid sequence and predicted 3D structure of Tg_chitinase from <i>T</i>. <i>gondii</i>.
<p>Tg_chitinase from <i>T</i>. <i>gondii</i> was identified by mass spectrometry using the amino acid sequences of tryptic peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144507#pone.0144507.t002" target="_blank">Table 2</a> (indicated in bold). (A) The amino acid sequence obtained from analysis of the peptide (SEIYQGDSVR) identified by BLASTP <i>T</i>. <i>gondii</i> GT1 (TGGT1_286465). (B) Predicted 3D structure of Tg_chitinase using I-TASSER.</p
Chitinase activity from <i>T</i>. <i>gondii</i>.
<p>Soluble <i>T</i>. <i>gondii</i> antigens were purified by affinity with an IgY-Sepharose 4B column, and the chitinase activity of the delayed fractions was measured. (A) Electrophoresis analysis of purified chitinase from <i>T</i>. <i>gondii</i>. First lane—MW (Molecular Weight Ladder), second lane—purified chitinase in 12% SDS-PAGE. (B) Crude extract and purified chitinase activity from <i>T</i>. <i>gondii</i> was measured. Error bars represent standard errors calculated from three replicates.</p
Tg_chitinase subcellular localization.
<p>(A) <i>T</i>. <i>gondii</i> tachyzoite DNA was stained with DAPI (blue), and (B) immunostained for Tg_chitinase with an anti-chitinase antibody, which was biotinylated with an antibody conjugated to anti-streptavidin-FITC (green). Superimposition of images stained for Tg_chitinase and DAPI (Merge and phase microscopy, C and D, respectively). Tg_chitinase is localized throughout the cytosol of <i>T</i>. <i>gondii</i> tachyzoites. Scale bar = 2.5 μm.</p