Proteases of Scedosporium aurantiacum, an opportunistic fungal pathogen

Scedosporium aurantiacum is an opportunistic filamentous fungal pathogen that is capable of causing a range of infections in immunocompromised people. In Australia, S. aurantiacum is the second most common fungus associated with cystic fibrosis after Aspergillus fumigatus. The majority of the work carried out with S. aurantiacum currently described in the literature features prevalence studies, animal studies including virulence and antifungal susceptibility studies. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. This work aimed at profiling and identifying proteases secreted by S. aurantiacum with a view to investigating a potential link with protease production and fungal pathogenicity. Clinical (WM 06.482) and environmental (WM 10.136) S. aurantiacum isolates were grown in a synthetic cystic fibrosis sputum medium supplemented with either casein supporting the synthesis and secretion of a wide range of proteases or mucin, which mimicked the cystic fibrosis lung sputum. Liquid cultivations were performed under both normoxic and hypoxic conditions considering the fact that fungi have to encounter oxygen level differences between the environment and human organs. Secreted proteases in the culture supernatants were examined using enzymatic assays with class-specific substrates, inhibition assays, zymogram activity analysis and protein identification by mass spectrometry. Serine proteases from both strains were responsible for the majority of protease activity under normoxia; elastase- and trypsin-like proteases from the clinical isolate had significantly higher activities than those from the environmental isolate. Under hypoxia, secreted proteases from both isolates were predominated by the aspartic class; cysteine proteases were also detected. The clinical isolate had higher acidic protease activity including aspartic and cysteine protease activities, while the environmental isolate had higher alkaline protease activity featuring chymotrypsin-, subtilisin-, elastase- and trypsin-like protease activities. Protein identification was conducted for both isolates grown under normoxia and hypoxia. Three protease homologs were identified with high sequence similarity to known fungal proteases under normoxia including subtilisin protease S8, putative leucine aminopeptidase and a PA-SaNapH-like protease. Aspartic protease homologs were identified under hypoxia. Cysteine proteases secreted by the clinical S. aurantiacum isolate under hypoxia were involved in both detachment and death of the A549 human lung epithelial cells in vitro, causing rounding up of the cells and release of the cell contents into the environment. Chymotrypsin-like protease from hypoxic cultures was also involved in cell detachment by rounding cells up, yet the floating cells were still alive. Elastase-like protease from normoxic cultures was found to cause cell death by a yet unidentified mechanism. This work expands the existing knowledge of the emerging fungal pathogen S. aurantiacum and provides insights into roles of secreted proteases in fungal invasion.