title: Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner.
abstract: 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.
authors.affiliation: 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.
authors.forename: Priscila
authors.initials: P
authors.lastname: de Oliveira
authors.affiliation: 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.
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.affiliation: 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.
authors.forename: Aparecida Sadae
authors.initials: AS
authors.lastname: Tanaka
authors.affiliation: 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.
authors.forename: Adriana Karaoglanovic
authors.initials: AK
authors.lastname: Carmona
authors.affiliation: 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.
authors.forename: Saara Maria Batista
authors.initials: SM
authors.lastname: Dos Santos
authors.affiliation: 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.
authors.forename: Bianca Carla Silva Campitelli
authors.initials: BC
authors.lastname: de Barros
authors.affiliation: 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.
authors.forename: Paloma Korehisa
authors.initials: PK
authors.lastname: Maza
authors.affiliation: 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.
authors.forename: Rosana
authors.initials: R
authors.lastname: Puccia
authors.affiliation: 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. erika.suzuki@unifesp.br.
authors.forename: Erika
authors.identifier: http://orcid.org/0000-0002-4010-8716
authors.initials: E
authors.lastname: Suzuki
mesh_terms: D000072283:A549 Cells; D002460:Cell Line; D002470:Cell Survival / Q000276:immunology; D016207:Cytokines / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D010228:Paracoccidioides / Q000201:enzymology / Q000276:immunology; D010229:Paracoccidioidomycosis / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects; D044462:Receptors, Proteinase-Activated / Q000378:metabolism*
chemical_list: D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D010455:Peptides; D011480:Protease Inhibitors; D044462:Receptors, Proteinase-Activated; D010450:Endopeptidases
keywords: Cytokine; Epithelial cell; Fungus; Paracoccidioides; Protease; Protease-activated receptors