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

• Published in: Revista iberoamericana de micología 2018-01, Vol.35 (1), p.32-38
• Main Authors: Sabanero López, Myrna, Flores Villavicencio, Lérida L., Soto Arredondo, Karla, Barbosa Sabanero, Gloria, Villagómez-Castro, Julio César, Cruz Jiménez, Gustavo, Sandoval Bernal, Gerardo, Torres Guerrero, Haydee
• Format: Article
• Language: English
• Subjects: Citoesqueleto; Cytoskeleton; Epitelio; Epithelium; Interacción; Interactions; Proteasas; Proteases; Sporothrix schenckii
• ISSN: 1130-1406
• DOI: 10.1016/j.riam.2017.05.003

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. To evaluate the proteolytic activity of S. schenckii on epithelial cells. 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. 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. 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. 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. Evaluar la actividad proteolítica de S. schenckii en células epiteliales. 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. 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 act