Proteases of Sporothrix schenckii: cytopathological effects on a host-cell model / Proteasas de Sporothrix schenckii: efectos citopatológicos en un modelo de células huésped

Background. Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. Aims. To evaluate the proteolytic activity of S. schenckii on epithelial cells. Methods. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. Results. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Conclusions. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors (AU)

Antecedentes. La esporotricosis es una infección fúngica causada por el complejo Sporothrix schenckii. La adhesión del hongo al tejido hospedero se ha considerado un paso clave en la colonización e invasión, sin embargo poco se conoce de los eventos tempranos en la interacción hospedero-parasito. Objetivos. Evaluar la actividad proteolítica de S. schenckii en células epiteliales. Métodos. El sistema proteolítico (bajo los valores pH 5 y 7) fue evaluado mediante azocoll y zimogramas. Además, la interacción hospedero-parasito y la respuesta celular fueron analizadas con el examen de los microfilamentos del citoesqueleto mediante faloidina-FITC y microscopia electrónica de transmisión. Finalmente, la actividad metabólica (viabilidad celular) fue determinada por un ensayo de XTT. Resultados. Los zimogramas de S. schenckii muestran que posee una alta actividad proteolítica intracelular y extracelular (Mr≥200, 116, 97 y 70kD) dependientes de pH e inhibidas por PMSF y E64, que actúan sobre serin- y cistein proteasas. Durante la interacción de las células epiteliales-proteasas, las células mostraron alteraciones en la distribución de los microfilamentos y la estructura de la membrana plasmática. Además, la actividad metabólica (viabilidad celular) de las células epiteliales disminuyó un 60% sin inhibidores de proteasas. Conclusiones. Nuestros datos demuestran la complejidad de la respuesta celular durante el proceso de infección, proceso que puede ser en parte contrarrestado por la acción de los inhibidores de proteasas. Además, los resultados proporcionan información crítica para el entendimiento de la naturaleza en la interacción hospedero-hongo y para una nueva terapia antifúngica eficaz que incluya inhibidores de proteasas (AU)

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

BACKGROUND: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. AIMS: To evaluate the proteolytic activity of S. schenckii on epithelial cells. METHODS: The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. RESULTS: The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. CONCLUSIONS: Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors.

Cell wall glycoproteins participate in the adhesion of Sporothrix schenckii to epithelial cells.

Sporothrix schenckii is the etiologic agent of sporotrichosis. This fungal infection is an emerging disease potentially fatal in immunocompromised patients. The adhesion to host cells is a crucial early event related with the dissemination of pathogens. In order to clarify the mechanisms of adhesion of S. schenckii yeast cell to epithelial cells, we studied the biochemical basis of this process. The electrophoretic analysis of cell wall protein from S. schenckii coupled at ConA and stained with HRP, revealed nine different proteins with MW ≥ 180, 115, 90, 80, 58, 40, 36, 22 and 18 kDa. Using ligand-like assay with biotinylated S. schenckii surface proteins, five proteins with MW ≥ 190, 180, 115, 90 and 80 kDa which have affinity to epithelial cells were identified. The adhesion of yeast to epithelial monolayer was significantly inhibited when S. schenckii was pretreated with concanavalinA (ConA) and wheat germ agglutinin (WGA) lectins, alkali, periodate, trypsin, endoglycosidase H (EndoH), salt solutions and detergents. The ability of adhesion of S. schenckii yeast was recovered by blocking the lectin with sugar complementary. These data suggest that surface glycoprotein with mannose and glucose residue could be participate in the process of fungal adhesion to epithelial cells.