1 3 Med Microbiol Immunol DOI 10.1007/s00430-016-0490-x ORIGINAL INVESTIGATION Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner Priscila de Oliveira 1 · Maria Aparecida Juliano 2 · Aparecida Sadae Tanaka 3 · Adriana Karaoglanovic Carmona 4 · Saara Maria Batista dos Santos 2 · Bianca Carla Silva Campitelli de Barros 1 · Paloma Korehisa Maza 1 · Rosana Puccia 5 · Erika Suzuki 1 Received: 13 July 2016 / Accepted: 8 December 2016 © Springer-Verlag Berlin Heidelberg 2016 fungal culture supernatants in a p-aminomethylbenzami- dine-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, incu- bation 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. bra- siliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion. Keywords Protease-activated receptors · Epithelial cell · Fungus · Paracoccidioides · Protease · Cytokine Introduction Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermally dimorphic fungi that are endemic in Latin America, mostly in Brazil. These pathogens cause paracoc- cidioidomycosis (PCM), a human systemic mycosis, often characterized by a slow and progressive disease, predomi- nantly affecting the lungs and mucocutaneous tissues. The fungus may eventually disseminate throughout the patient’s body, infecting the central nervous tissues, mucosa, adrenal glands, skin, lymphoid tissue, or any other organs [1, 2]. Proteases are virulence factors in fungal infectious pro- cesses. Among different pathogenic mechanisms, these enzymes may damage the host tissues (e.g., skin, airway epithelium) and/or inactivate several immune system mol- ecules, such as immunoglobulins or components of the Abstract Paracoccidioides brasiliensis is one of the etio- logical agents of the human systemic mycosis paracoc- cidioidomycosis. 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 path- ogenic 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 Electronic supplementary material The online version of this article (doi:10.1007/s00430-016-0490-x) contains supplementary material, which is available to authorized users. * Erika Suzuki erika.suzuki@unifesp.br 1 Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina - Universidade Federal de São Paulo, Rua Botucatu, 862 – Ed. Antônio C. M. Paiva – 6 andar, São Paulo, SP 04023-062, Brazil 2 Department of Biophysics, Escola Paulista de Medicina - Universidade Federal de São Paulo, Rua Três de Maio, 100 – INFAR – 1 andar, São Paulo, SP 04044-020, Brazil 3 Department of Biochemistry, Escola Paulista de Medicina - Universidade Federal de São Paulo, Rua Três de Maio, 100 – INFAR – 5 andar, São Paulo, SP 04044-020, Brazil 4 Department of Biophysics, Escola Paulista de Medicina - Universidade Federal de São Paulo, Rua Pedro de Toledo, 699 – 7 andar, São Paulo, SP 04039-032, Brazil 5 Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina - Universidade Federal de São Paulo, Rua Botucatu, 862 – Ed. Antônio C. M. Paiva – 8 andar, 04023-062 São Paulo, SP, Brazil