Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Interação de Paracoccidioides brasiliensis com células endoteliais / Interaction of Paracoccidioides brasilienses with endothelial cells

A paracoccidioidomicose apresenta um amplo espectro de manifestações clínicas e Paracoccidioides brasiliensis, seu agente etiológico, pode atingir vários tecidos com ênfase ao pulmão. A migração de fungos patogênicos através da camada de células endoteliais é considerada pré-requisito para a invasão de múltiplos órgãos e sua disseminação. No presente estudo verificou-se a adesão de P. brasiliensis às células endoteliais in vitro e se esta adesão poderia representar um mecanismo para a disseminação do fungo. Para tanto, além da técnica convencional de microscopia ótica, uma outra metodologia foi desenvolvida, emblocando os cordões umbilicais em parafina, no intuito de detectar o fungo presente no material (in vivo). Experimento de migração de P. brasiliensis através da monocamada de células endoteliais também foi realizado, e nos poços sem células, a migração de células leveduriformes foi maior em menor período de tempo. Os fungos conseguiram passar através da monocamada, quando comparados com o controle sem as células, mas com redução em torno de 30%. Isso mostra que a monocamada foi parcialmente impediente para o fungo, mas que este foi capaz de migrar através dessas células. Em nossos experimentos com estas células, houve grande dificuldade de se encontrar P. brasiliensis aderido ao tapete celular nos períodos de tempo padronizados. Sugere-se com esses resultados que o fungo atravessa as células endoteliais de uma maneira muito rápida, que não pode ser detectada através do cultivo in vitro. Portanto, P. brasiliensis teria capacidade de atravessar rapidamente as células endoteliais e provavelmente alcançar tecidos mais profundos

Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells.

Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P. brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin.Very little is known about early interactions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P.brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P. brasiliensis were compared, and strain 18 (high virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M (mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85% of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis.

Pathogenesis II: fungal responses to host responses: interaction of host cells with fungi.

Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures. The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules. Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival. Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity. Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules. The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization. Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic. It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation. For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence. Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell. These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface. The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.