Aspartic Proteases Drive Stress Response, Morphogenesis, and Macrophage Interaction in Sporothrix brasiliensis

Sporotrichosis, a globally distributed subcutaneous mycosis, finds its most virulent agent in the species Sporothrix brasiliensis, particularly in zoonotic outbreaks associated with cats. This study investigated the role of aspartic proteases (APs) as potential virulence factors in S. brasiliensis. Through in silico analysis, we identified and characterized 19 genes encoding putative aspartic proteases in the S. brasiliensis genome. Susceptibility assays to different stressors demonstrated distinct profiles between the Sb1168 and Sb5110 strains, but the presence of Pepstatin A strongly inhibited growth under stress, indicating a crucial role for these enzymes in environmental adaptation. Proteolytic activity was modulated by cell wall stressors. Strain Sb1168 showed higher aspartic protease activity (87% inhibition by pepstatin A), while Sb5110 exhibited a mixed enzymatic profile, with significant contributions from metalloproteases (50% inhibition by EDTA and pepstatin A individually, and 80% in combination). Inhibition of APs blocked the mycelium-to-yeast (M → Y) dimorphic transition, suggesting that these proteases are dimorphism regulators. In macrophage assays, APs inhibition resulted in a significant increase in the phagocytic index and a reduction in intracellular fungal viability (CFU count), in addition to altering the profile of secreted cytokines (increase in pro-inflammatory IL-12 and decrease in anti-inflammatory IL-10 in treated Sb1168), suggesting these enzymes modulate immune evasion.