Navegando por Palavras-chave "Cell Wall"

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    Avaliação do papel da hipóxia na modulação da parede celular do fungo Paracoccidioides brasiliensis
    (Universidade Federal de São Paulo, 2021-09-24) Barros, Yasmin Nascimento de [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; http://lattes.cnpq.br/4373797404389169; http://lattes.cnpq.br/6980283990020398
    Fungos termodimórficos do gênero Paracoccidioides são os agentes etiológicos da paracoccidioidomicose (PCM), uma micose sistêmica com ocorrência na América Latina. A interação entre Paracoccidioides spp. e seu ambiente extracelular, são de grande importância para o sucesso do estabelecimento do fungo no hospedeiro. Embora os fungos patogênicos respondam a diferentes estresses, tais como: altas temperaturas, ROS/RNS, alterações de pH e limitação de macro e micronutrientes, pouco se conhece sobre como os fungos patogênicos humanos respondem à limitação de oxigênio, particularmente quando condições hipóxicas são encontradas no tecido hospedeiro. Neste sentido, o objetivo do trabalho é avaliar a modulação dos componentes da parede celular de Paracoccidioides brasiliensis em respostaa ambientes com baixas concentrações de oxigênio. Os resultados demonstram um aumento viabilidade do P. brasilienses quando incubado a baixas concentrações de oxigênio. Além disso, genes envolvidos na síntese de α-glucana aumentaram ao mesmo tempo que genes relacionados a síntese de β-glucana reduzem sua expressão relativa em resposta a hipóxia. Ensaio de interação entre β-glucana e Dectina-1 por citometria de fluxo demonstrou que em baixas concentrações de oxigênio, há uma redução da interação entre β-glucana e seu receptor. Além disso, Leveduras de P. brasiliensis apresentam uma resistência à fagocitose quando submetido à hipóxia. Em adição, a via de cAMP-PKA não parece estar envolvida na modulação dos componentes de parede celular de leveduras de P. brasiliensis sob baixas concentrações de oxigênio. Por fim, as leveduras submetidas a hipóxia apresentaram uma menor suscetibilidade ao antifúngico fluconazol. Em conjunto, os resultados encontrados nesse trabalho sugerem uma modulação da parede celular do fungo patogênico P. brasiliensis em resposta à hipóxia.
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    Estudo do papel da Histidina quinase reguladora do dimorfismo (DRK1) do fungo patogênico Paracoccidioides brasiliensis
    (Universidade Federal de São Paulo (UNIFESP), 2021) Navarro, Marina Valente [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; Universidade Federal de São Paulo
    Thermal dimorphic fungi of Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), a systemic endemic disease in Latin America with high incidence in Brazil. This pathogen presents as infective mycelium at 25°C in the soil, reverting to its pathogenic form when inhaled by mammalian host (37°C). This ability to switch from mycelium to yeast form is essential to disease development. Among dimorphic fungus species, dimorphism regulating histidine kinase (Drk1) plays an essential role to morphological transition. Histidine kinases (HK) are very important as virulence and cellular survival regulators. These kinases are present in bacteria and fungi, but absent in mammalian cells. This evidence suggests that HKs are interesting pharmacological targets to fungal diseases. Recently, our group characterized P. brasiliensis DRK1 (PbDRK1) expression during mycelium-yeast transition. It was observed that this gene is predominantly expressed in pathogenic yeast phase. In addition, we verified that when Dkr1 pharmacological inhibitor (iDrk1) was incubated with mycelium at 37°C, it alters the dimorphic switch. Hence, the purpose of this study was to investigate the role of PbDrk1 in cell wall modulation of P. brasiliensis. Previous data indicates a correlation between PbDrk1 and MAP kinase Hog1 over osmotic stress condition. In the present work it was observed decreased Hog1 phosphorylation levels after incubation with iDrk1 over osmotic stress but also related with cell wall integrity, confirming the participation of this HK in Hog1 signaling pathway. Additionally, we observed that PbDrk1 participates in fungal resistance to different cell wall disturbing agents, such as Congo Red, Calcofluor White and sodium chloride, by reducing yeasts viability after treatment with iDrk1. In order to verify the role of PbDRK1 in cell wall morphogenesis, we performed RTqPCR analysis. The results indicated that samples previously exposed to iDrk1 presented higher expression levels in several genes related to cell wall modulation. Among the genes analyzed, we highlighted FKS1, a β-glucan synthase which was 3,6-fold increase when compared to no treatment control. Confocal microscopy analysis and flow cytometry showed higher β-glucan exposure in cell surface of P. brasiliensis after 24 h incubation with iDrk1. Accordingly, through phagocytosis assay, it was observed a significant higher phagocytic index in yeasts treated with iDrk1 than control group, evidencing the role of PbDrk1 in cell wall modulation, which becomes a relevant target to be investigated. In parallel, the immune response profile was also assayed, with increased levels of pro-inflammatory cytokines. It is important to highlight that iDrk1 concentration used on this study did not reduce yeast viability. Finally, our data strongly suggests that PbDrk1 modulates cell wall components expression,among which we can point out b-glucan. The comprehension of this signaling pathway may be of great value to identify targets to antifungal molecular activity, since HK are not present in mammals.