IL‐6 Degradation by Secreted Proteases From <i>Paracoccidioides restrepiensis</i>

Paracoccidioidomycosis is a systemic fungal disease caused by <i>Paracoccidioides</i> spp., predominantly affecting populations in Latin America, with Brazil reporting the highest number of cases. The infection is associated with severe pulmonary and systemic manifestations. Previous studies have highlighted the role of fungal proteases in adhesion, invasion, and the modulation of host immune responses, implicating them as key virulence factors. Our group previously demonstrated that <i>Paracoccidioides restrepiensis</i> secretes proteases that activate protease-activated receptors (PAR-1 and PAR-2) in human lung epithelial cells, stimulating the secretion of proinflammatory cytokines, including IL-6 and IL-8. We hypothesized that <i>P. restrepiensis</i> secretes proteases that are capable of degrading key host cytokines, such as IL-6, thereby contributing to modulate the host immune response during infection. This study is aimed at identifying and characterizing proteases secreted by <i>P. restrepiensis</i> that degrade human IL-6. Proteases secreted by <i>P. restrepiensis</i> were isolated using a <i>p</i>-aminomethylbenzamidine (<i>p</i>ABA)-Sepharose affinity column. Protease-containing fractions were incubated with recombinant human IL-6 and further analyzed by Western blot to evaluate their ability to degrade this cytokine. Fractions were submitted to liquid chromatography and mass spectrometry to characterize the proteome content, focusing on the identification of fungal proteases. The hydrolysis of IL-6 in the presence of different protease inhibitors was also analyzed to confirm the specific activity of the fungal proteases. Enzymatic assays revealed proteases that hydrolyze human IL-6, suggesting a mechanism by which <i>P. restrepiensis</i> modulates the host immune response. In addition, mass spectrometry analysis confirmed the presence of a serine protease in the protease activity-containing fractions. These findings indicate that <i>Paracoccidioides</i> proteases may modulate host immune response by degrading key cytokines involved in inflammation and host defense.