Elucidating the virulence roles of secreted aspartyl proteinase (Sap 1972) in non-albicans Meyerozyma guilliermondii through construction of a structure-guided mutant

Meyerozyma guilliermondii (Candida guilliermondii) is one of the non-albicans Candida species associated with invasive candidiasis. M. guilliermondii strain SO that was isolated from spoiled orange could express industrially important enzymes without hazardous methanol as inducer. Our prior studies confirmed the presence, expression, and activity of virulence-associated secreted aspartyl proteinases (MgSap). Given the time-consuming process to delete all MgSAP members from the fungal genome, this study aimed to elucidate the virulence roles of MgSap by constructing a structure-guided mutant. As the most conserved MgSap with pathogenic Candida Sap, MgSap1972 was predicted to possess the largest druggable active cleft (1122.652 Å2) despite the undruggable cleft in MgSap4979 (1323.165 Å2). Employing the homologous recombination strategy, we successfully deleted MgSAP1972 from the fungal genome. The resulting mutant (MgSOΔSAP1972) showed slightly heightened susceptibility to LiCl and exhibited a 38.8 % reduction in biofilm mass than the wild type. With the normal heartbeat and slightly higher hatching rates, a 16.7 % decrease in the mortality rate of zebrafish embryos at 96 h post-exposure was observed, where several sub-lethal endpoints such as kyphosis and short tail were absent in embryos infected with MgSOΔSAP1972. These findings underscore the virulence roles of MgSap1972 in the fungal pathogenicity of M. guilliermondii strain SO. Our study showcases an efficient in silico-in vitro-in vivo pipeline for identifying and targeting fungal virulence factors, facilitating hypothesis construction prior to validation through mutant generation. This investigation offers significant understanding of potential targets for therapy, establishing a foundation for future efforts to combat C. guilliermondii infection.

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