Aspartic Protease Inhibition Induces Proteomic Remodeling in Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a major systemic mycosis in Latin America caused by Paracoccidioides brasiliensis, yet the contribution of aspartic proteases to fungal physiology and pathogenicity remains poorly understood. Here, we employed data-independent acquisition (DIA)-based quantitative proteomics to investigate the impact of pepstatin A-mediated inhibition of aspartic proteases on the P. brasiliensis proteome. High-coverage analysis identified up to 21,156 peptides and approximately 3000 proteins per condition, revealing progressive and time-dependent proteomic remodeling after inhibitor exposure. Differential abundance analysis demonstrated changes in proteins associated with membrane organization, protein turnover, RNA metabolism, stress adaptation, and cellular homeostasis. Functional enrichment analyses indicated modulation of metabolic processes, oxidative phosphorylation-associated proteins, and other biological functions related to fungal adaptation. Proteins associated with autophagy-related processes were differentially regulated, while MDC staining revealed temporal changes in acidic vesicle formation, suggesting alterations in vesicle-associated responses. Additionally, pepstatin A treatment reduced reactive oxygen species (ROS) production, consistent with changes in proteins related to oxidative metabolism. The inhibitor also impaired fungal adhesion to A549 pulmonary epithelial cells, indicating that aspartic proteases may influence host-pathogen interactions through mechanisms beyond adhesin abundance alone. Collectively, these findings provide a systems-level view of the cellular responses induced by aspartic protease inhibition and highlight the importance of these enzymes in regulating P. brasiliensis adaptation, homeostasis, and pathogenic potential.