Navegando por Palavras-chave "Fungus"

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    Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner
    (Springer, 2017) de Oliveira, Priscila [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Batista dos Santos, Saara Maria [UNIFESP]; Silva Campitelli de Barros, Bianca Carla [UNIFESP]; Maza, Paloma Korehisa [UNIFESP]; Puccia, Rosana [UNIFESP]; Suzuki, Erika [UNIFESP]
    Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.
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    Seleção de fungos filamentosos como eficientes fontes de lignina-peroxidases
    (Universidade Federal de São Paulo (UNIFESP), 2020-08-21) Lima, Lidiane Maria dos Santos [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São Paulo
    Selection of Filamentous Fungi as Efficient Sources of Lignin-Peroxidases Lignin peroxidases (LiP) are ligninolytic enzymes normally produced by biodegradable fungi of lignocellulose in nature. Currently, interest in such enzymes has increased, due to their high potential for biotechnological application. In this sense, the search for microbial cultures, of different habitats, with different environmental conditions, exponentially increases the acquisition of notable enzymes for the most diverse purposes. An example is the application of biorefineries of cellulosic ethanol, which aims at the efficient transformation of lignocellulosic biomass into bioproducts, such as biofuels and energy, thus being considered a sustainable alternative to petroleum derivatives. However, one of the obstacles to the lignocellulose valorization on an industrial scale is the complexity and recalcitrance of biomass, especially lignin. It is in this context that the development and optimization of enzymatic preparations based on lignin-peroxidases are required, in order to enable the cleavage and digestibility of lignocellulosic fiber. Thus, the present study was initiated by the screening of environmental filamentous fungi with respect to the effectiveness of lignin-peroxidases, using dye degradation tests of aromatic molecular structures, such as remazol brilliant blue R (RBBR) and methylene blue (AM), as compounds for detecting fungal ligninolytic activity. Thus, six (6) filamentous fungi FPZSP3_01, FPZSP3_05, FPZSP3_47, FPZSP3_91, FPZSP1_129 and FPZSP1_141 isolated from soil increased by filter cake from sugar and alcohol refineries, were selected. Thus, such microorganisms were subjected to in-depth analysis about the enzymatic kinetics of their lignin-peroxidases, analyzing the crude enzymatic extract (microbial supernatant) from the following strains: FPZSP3_47 (Mucor sp.), FPZSP1_129 (Byssochlamys nivea) and FPZSP1 (Paecilomyces saturatus). The apparent kinetic constants KM, Vmax and kcat were determined in two conditions, pH 3.0 at room temperature (20 ºC±2), and pH 9.0, NaCl 4M, at 65 ºC, in addition to the influence of different temperatures, pH ranges and salinities in the activities enzyme detected. The filamentous fungus Mucor sp. (FPZSP3_47) belonging to the phylum Mucoromycota was selected for protein characterization studies. The apparent kinetic constants KM (mM) 55.65 ± 6.56, Vmax (mmol min-1) 414.75 ± 6.37 and kcat (min-1) 7.45 were higher at pH 3.0 at room temperature for Mucor sp. FPZSP3_47. The lignin-peroxidases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus exhibited activity and stability for 30 hours at all temperatures evaluated (4 ºC, ambient, 30 ºC and 65 ºC), with two optimal temperatures (30 ºC and 65 ºC) being selected. ºC). Such conditions were selected to analyze the activity dependence and enzymatic stability at pH. All ligninases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus were active and stable over a wide pH range (5.0 to 9.0). The pH 9.0 and the temperature of 65 ºC were selected to investigate the effect of NaCl (0.1 to 4 M) on the stability of LiP, and the results revealed wide tolerance to NaCl, for 24 hours. The LiP of the isolate Mucor sp. was purified by ion-exchange chromatography and analyzed on SDS-PAGE 12%, featuring a protein with a molecular mass estimated at 60 kDa and specific activity of 0.013 U/mg. It is worth mentioning that this is the first description of ligninases of a fungus of the genus Mucor sp. in the literature, therefore, a potential candidate for the optimization of processes associated with the production of ethanol from vegetable biomass.