Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.
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We thank Alisson Leonardo Matsuo for technical support with PbP isolation. This work was supported by the São Paulo Research Foundation (FAPESP Grants 2011/23096-8, 2011/22773-6, and 2015/25652-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES AUX PE PNPD Grant 2386/2011), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grants 303357/2012-6 and 306163/2015-2).
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de Oliveira, P., Juliano, M.A., Tanaka, A.S. et al. Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner. Med Microbiol Immunol 206, 149–156 (2017). https://doi.org/10.1007/s00430-016-0490-x
- Protease-activated receptors
- Epithelial cell