Navegando por Palavras-chave "Fungos patogênicos"

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    Caracterização de epitopos contendo resíduos de alfa-galactopiranosil em Paracoccidioides e outros fungos patogênicos
    (Universidade Federal de São Paulo (UNIFESP), 2019-08-29) Gegembauer, Gregory [UNIFESP]; Puccia, Rosana [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The main focus of this work was to find αGal epitopes (terminal α-galactopisyl) in fungi of medical importance, considering that there is little information regarding the possible roles of these glycotopes in pathogenic fungal systems. Our project aimed at expanding the group’s reported observation that yeast cells from Paracoccidioides brasiliensis Pb18 reacted with anti-αGal antibodies from chagasic patients (Ch-anti-αGal) on the cell wall and inside intracellular vacuoles. Those results were obtained during the characterization of αGal epitopes in extracellular vesicles produced by P. brasiliensis Pb18 and Pb3 using Ch-antiαGal and anti-αGal IgGs isolated from paracoccidioidomycosis patients, as well MOA lectin specific to Galα1,3Gal disaccharide. Our present confocal microscopy results using Ch-anti-αGal antibodies suggested the presence of αGal epitopes in Pb18, Pb3, Pb01, Histoplasma capsulatum, Candida albicans, Cryptococcus capsulatum, and Aspergillus fumigatus. The reactivity was visualized inside vacuoles and on the cell wall of mother cells and buds, especially at bud scar and bud necks. C. neoformans yeasts were labeled only at the cell wall in our experimental conditions. Ch-anti-αGal antibodies were also localized in the filamentous phase of Pb18 and H. capsulatum, mostly in the hyphae cytoplasm. In A. fumigatus, label predominated at the phialides surface. During the Pb18 mycelium to yeast phase, Ch-antiαGal antibodies intensely labeled transition round structures, thus suggesting a role in yeast formation. Additionally, the presence of αGal epitopes in the studied fungal species was strongly suggested by the effect of α-galactosidase treatment, which evoked intense cell aggregation. Altogether these results suggest the presence of αGal epitopes in several pathogenic fungi, both in the yeast and mycelium phases. An extended in silico analysis using fungal data banks has shown the presence of potential αgalactosyltransferase genes in all fungal species analyzed in this study, in view of the sequence homology with α-galactosyltransferases (αGTs) well described in Schizosaccharomyces pombe. We used as templates the sequences of seven α1,2- and three α1,3-galactosiltransferases. There were similar sequences to α1,2-GT GMH4 and GMH5 (GT34 family), as well to OTGs α1,3-GT (GT8 family). The gene expression of related genes was analyzed between mycelium and yeast phases. The RT-PCR results suggest, in our conditions, that the Pb01 GMH5-like gene was more transcribed than that from Pb3 and Pb18 in both phases. The GMH-like genes were considerably more expressed during the yeast phase in all isolates than in the mycelium phase. These results suggest that there are potential αGT genes being expressed in P. brasiliensis and P. lutzii, however the mRNA accumulation varies with the isolate, cell phase and culture conditions. The overall data presented in this thesis are totally original in the field of pathogenic fungi and upgrade the knowledge about αGal epitopes, which can potentially have important roles in pathogenesis of mycosis, thus mirroring what happens in Chaga’s disease.
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    CARACTERIZAÇÃO ESTRUTURAL E IMUNOQUÍMICA DE GLICOINOSITOL FOSFORILCERAMIDAS DE Aspergillus fumigatus. EFEITO DE INIBIDORES DE SÍNTESE DE GLICOESFINGOLIPÍDEOS EM FUNGOS PATOGÊNICOS
    (Universidade Federal de São Paulo (UNIFESP), 2006-01-01) Guimarães, Luciana Lopes [UNIFESP]; Takahashi, Helio Kiyoshi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The search for alternatives for new antifungal therapies led our laboratory to analyze the molecular structures of specific glycosphingolipids (GSLs) present in fungi but not in mammals. We analyze the molecular structures of glycosylinositol phosphorylceramides (GIPCs) from the opportunistic fungus Aspergillus fumigatus. GSLs were extracted and purified by a combination of ion exchange chromatography (DEAE-Sephadex), High Performance Liquid Chromatography (HPLC) and preparative High Performance Thin Layer Chromatography (HPTLC). The structures of the GIPCs from A. fumigatus were elucidated by GC/MS, and were described as: Af-2: Manpα1→3Manpα1→2InsPCer Af-3a: Galfβ1→6Manpα1→3Manpα1→2InsPCer Af-3b: Manpα1→3(Galfβ1→6)Manpα1→2InsPCer Af-3c: Manpα1→3Manpα1→6GlcpNα1→2InsPCer Af-4: Galfβ1→6Manpα1→3(Galfβ1→6)Manpα1→2InsPCer By HPTLC immunostaining, Af-3a, Af-3b and Af-4 were found to be reactive with sera of patients with aspergillosis, probably the Galf terminal residue present in these structures would represent the antigenic component recognized by the antibodies present in these sera. This is the first description of the glycan structures displayed in the GIPCs Af-3a and Af-4. The structures from Af-2 and Af-3b were previously described in P. brasiliensis (Pb-2 and Pb-1, respectively). Af-3c was previously described in S. schenckii (Ss-Y6). These results point to new directions, that could lead to the development of more efficient methodologies in the immunodiagnostic of aspergillosis, increasing the specificity of the current protocols. To better understand the importance/biological role of GSLs in different fungi, we tested inhibitors of GlcCer synthase and IPC synthase in cultures of pathogenic fungi. The addition of the inhibitor of GlcCer synthase, D-P4, was efficient in the inhibition of the growth in all of the species of fungi studied in this thesis: Paracoccidioides brasiliensis, Histoplasma capsulatum, Sporothrix schenckii and Criptococcus neoformans. The inhibitor of IPC synthase, Aureobasidin A (AbA), was also efficient in inhibition of the growth of these fungi. AbA (40μM) inhibited 100% of the colony forming of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Criptococcus neoformans, however, in S. schenckii the same concentration only inhibited 50% of the colony forming. Either D-P4 or AbA were able to inhibit the yeast-mycelium transition in P. brasiliensis and H. capsulatum. The results presented here may open attractive perspectives for new antifungal therapies based on the specific inhibition of enzymes of the biosynthetic pathway of fungal GSLs.
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    Estudo da atividade da GTPaseRas de Paracoccidioides brasiliensis durante o termo-dimorfismo do fungo e após estresse induzido por espécies reativas de oxigênio e nitrogênio
    (Universidade Federal de São Paulo, 2017-03-30) Conceicao, Palloma Mendes [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Paracoccidioides brasiliensis, a thermally dependent dimorphic fungus, is one of the etiological agents of paracoccidioidomycosis (PCM). The ability to evade the innate immune response of the host and cause disease is particularly due to its ability to respond to and survive the nitrosative / oxidative stress caused by cells of the immune system. However, nothing is known about the regulation of signal transduction pathways associated with this resistance, possibly triggered by the reactive oxygen and nitrogen species generated by the host and that will act on this microorganism. Among the proteins that participate in cell signaling pathways, Ras is a GTPase with important role in different cellular processes, such as the control of morphological changes and cell proliferation. One of the main objectives of this work was to verify the activity of Ras under different study conditions. Initially it was observed that the fungus treated with Ras inhibitor and used to infect mice had a lower number of CFU in the lung when compared to the untreated control. These data suggest that Ras is important in fungal virulence. It has also been demonstrated that Ras is activated in response to oxidative, nitrosative and thermal stress. In addition, Ras-GTP participates in the transition from mycelial to yeast form. In this study it was also verified that Ras-GTP interacts with both Byr2 (Ste11) and Hog-1 in response to oxidative and nitrosative stress. Finally, it was verified that Ras can modulate the expression of some genes related to the response to oxidative and nitrosative stress. Therefore, this study presents a set of data indicating the presence of a (yet unknown) relationship between the Ras and Hog-1 signaling pathway in response to stress triggered by ROS / RNS, which may have been very important for the Response of P. brasiliensis to stress.
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    Papel das vesículas extracelulares no transporte de fatores de virulência em Paracoccidioides brasiliensis. Utilização do modelo Pb18 - variantes virulenta e atenuada.
    (Universidade Federal de São Paulo, 2022-05-06) Octaviano, Carla Elizabete [UNIFESP]; Puccia, Rosana [UNIFESP]; http://lattes.cnpq.br/8787784443554068; http://lattes.cnpq.br/8332956528895029
    Vesículas extracelulares (EVs) são componentes celulares arredondados delimitados por uma membrana de bicamada lipídico/proteica. Elas transportam para o ambiente extracelular, incluindo a parede celular, uma variedade de moléculas que potencialmente promovem sinalização à distância entre patógeno-hospedeiro e entre microrganismos. Sua importância na comunicação célula-célula, no remodelamento da parede celular e na virulência fúngica está começando a ser melhor explorada. A paracoccidioidomicose (PMC) é uma micose sistêmica de natureza granulomatosa crônica prevalente em áreas endêmicas na América Latina e é causada por espécies do fungo dimórfico dependente de temperatura Paracoccidioides spp. Nosso grupo e seus colaboradores têm se dedicado à caracterização de EVs deste e de outros fungos patogênicos. No presente trabalho, estudamos o efeito das EVs fúngicas utilizando o modelo do isolado Pb18 virulento/atenuado de P. brasiliensis. O modelo consiste em variantes que exibem transitoriamente maior (vPb18) ou menor (aPb18) patogenicidade, sendo que a virulência pode ser restabelecida por passagem em camundongos. Neste trabalho, a incubação de aPb18 com vEVs, isoladas de vPb18, foi capaz de reverter a sensibilidade de aPb18 ao estresse oxidativo e nitrosativo aos níveis apresentados por vPb18. Isso provavelmente ocorreu devido à expressão de moléculas antioxidantes, visto que houve aumento da expressão dos genes da oxidase alternativa AOX e das peroxirredoxinas HYR1 e PRX1, além de uma maior atividade de catalase. In vitro, aEVs (isoladas de aPb18) induziram uma produção significativamente maior de mediadores de inflamação, especificamente, óxido nítrico, TNF-α, IL-6 e MCP-1, quando co-incubadas com macrófagos da linhagem Raw 264.7 e macrófagos derivados da medula óssea, sendo que o efeito foi dose-dependente. In vivo, o tratamento subcutâneo com EVs (três doses, com intervalo de 7 dias entre as doses) antes do desafio com vPb18 exacerbou a PCM murina, conforme observado pelo maior número de unidades formadoras de colônias nos pulmões após 30 dias de infecção e pela análise histopatológica do tecido. Esse efeito foi mais pronunciado em camundongos tratados com aEVs, provavelmente porque as concentrações pulmonares de TNF- α, IFN-γ, IL-6 e MCP-1 estavam especialmente aumentadas nesses animais e evocaram uma resposta possivelmente hiperinflamatória. Nossos estudos foram realizados com EVs obtidas por ultracentrifugação diferencial de leveduras de ambas as variantes do isolado Pb18 cultivadas em meio definido Ham’s F-12 agar acrescido de 0,5% de glicose. Nessas condições, vEVs e aEVs apresentaram tamanhos semelhantes, mas provavelmente carregam conteúdos distintos, considerando que as vEVs tendem a agregar após armazenamento a 4°C e -20°C. Além disso, o conteúdo de proteína e ergosterol estavam significativamente dimunuídos em aEVs. Em conjunto, nossos resultados sugerem que EVs derivadas de vPb18 podem induzir em células receptoras aPb18 a expressão de traços de virulência característicos da variante virulenta. Os resultados apresentados nesta dissertação são originais e trazem uma contribuição importante para o entendimento sobre o papel das EVs fúngicas na comunicação célula-célula e sobre seu efeito na patogênese da PCM murina. Em nosso trabalho, esse efeito foi negativo, mas depende claramente das condições experimentais, uma vez que as EVs são estruturas complexas e dinâmicas.