Resumo

Proteases are important virulent factors found in different microorganisms. These enzymes are involved in the destruction of host tissues, causing cell damage and degradation of several molecules of the immune system, such as components of the complement system and cytokines. Recently, our group described that P. brasiliensis yeasts are able to induce expression of the inflammatory cytokines IL-6 and IL-8 in the human lung epithelial cells A549, and also, we verified that this fungus expresses proteases that are able to promote degradation of these cytokines. In the present work, we show that these proteases are secreted by yeast forms of P. brasiliensis. Next, human recombinant IL-6 (hrIL-6) was incubated in the presence of culture supernatants of yeast forms of P. brasiliensis strain 339 and several protease inhibitors (BmSI, a subtilisin inhibitor from Boophilus microplus; leupeptin, an inhibitor of serine and cysteine proteases; AEBSF and aprotinin, both inhibitors of serine proteases; p-HMB, a specific inhibitor of the serine-thiol proteases; pepstatin A, an inhibitor of aspartic acid proteases; EDTA, an inhibitor of metalloproteases; E-64, an inhibitor of cysteine proteases). Then, cytokine degradation was evaluated by SDS-Tricine PAGE and Western blot. We verified that hrIL-6 degradation was inhibited by by leupeptin, aprotinin, p-HMB and BmSI while other inhibitors did not have an effect on this proteolysis. For purification of these proteases, P. brasiliensis culture supernatants were applied to an affinity column of p-aminomethylbenzamidine (pABA)-Sepharose and ten fractions were obtained. We observed that the proteases capable to cleave hrIL-6 and hrIL-8 were eluted from this column, predominantly in fractions 3, 4 and 5.Therefore, taking together, these results suggest that yeasts of P. brasiliensis strain 339 secrete subtilisin-like serine proteases that degrade the human inflammatory cytokines IL-6 and IL-8, and in this manner, P. brasiliensis may modulate host inflammation. Supported by FAPESP, CNPq and CAPES.