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Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner

Medical Microbiology and Immunology volume 206, pages 149–156 (2017)Cite this article

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.

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References

  1. Ameen M, Talhari C, Talhari S (2010) Advances in paracoccidioidomycosis. Clin Exp Dermatol 35:576–580

    Article  CAS  PubMed  Google Scholar 

  2. Colombo AL, Tobón A, Restrepo A, Queiroz-Telles F, Nucci M (2011) Epidemiology of endemic systemic fungal infections in Latin America. Med Mycol 49:785–798

    PubMed  Google Scholar 

  3. Yike I (2011) Fungal proteases and their pathophysiological effects. Mycopathologia 171:299–323

    Article  CAS  PubMed  Google Scholar 

  4. Parente JA, Costa M, Pereira M, Soares CM (2005) Transcriptome overview of Paracoccidioides brasiliensis proteases. Genet Mol Res 4:358–371

    CAS  PubMed  Google Scholar 

  5. Carmona AK, Puccia R, Oliveira MC, Rodrigues EG, Juliano L, Travassos LR (1995) Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis. Biochem J 309(Pt 1):209–214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Puccia R, Carmona AK, Gesztesi JL, Juliano L, Travassos LR (1998) Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane. Med Mycol 36:345–348

    Article  CAS  PubMed  Google Scholar 

  7. Tacco BA, Parente JA, Barbosa MS, Báo SN, de Souza Gsóes T, Pereira M, Soares CM (2009) Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis. Med Mycol 47:845–854

    Article  CAS  PubMed  Google Scholar 

  8. Parente JA, Salem-Izacc SM, Santana JM, Pereira M, Borges CL, Bailão AM, Soares CM (2010) A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins. BMC Microbiol 10:292

    Article  PubMed  PubMed Central  Google Scholar 

  9. Hong JH, Hong JY, Park B, Lee SI, Seo JT, Kim KE, Sohn MH, Shin DM (2008) Chitinase activates protease-activated receptor-2 in human airway epithelial cells. Am J Respir Cell Mol Biol 39:530–535

    Article  CAS  PubMed  Google Scholar 

  10. Ossovskaya VS, Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84:579–621

    Article  CAS  PubMed  Google Scholar 

  11. Adams MN, Ramachandran R, Yau MK, Suen JY, Fairlie DP, Hollenberg MD, Hooper JD (2011) Structure, function and pathophysiology of protease activated receptors. Pharmacol Ther 130:248–282

    Article  CAS  PubMed  Google Scholar 

  12. Shin SH, Lee YH, Jeon CH (2006) Protease-dependent activation of nasal polyp epithelial cells by airborne fungi leads to migration of eosinophils and neutrophils. Acta Otolaryngol 126:1286–1294

    Article  CAS  PubMed  Google Scholar 

  13. Chiu LL, Perng DW, Yu CH, Su SN, Chow LP (2007) Mold allergen, Pen c 13, induces IL-8 expression in human airway epithelial cells by activating protease-activated receptor 1 and 2. J Immunol 178:5237–5244

    Article  CAS  PubMed  Google Scholar 

  14. Moretti S, Bellocchio S, Bonifazi P, Bozza S, Zelante T, Bistoni F, Romani L (2008) The contribution of PARs to inflammation and immunity to fungi. Mucosal Immunol 1:156–168

    Article  CAS  PubMed  Google Scholar 

  15. Cheng SC, Chai LY, Joosten LA, Vecchiarelli A, Hube B, Van Der Meer JW, Kullberg BJ, Netea MG (2010) Candida albicans releases soluble factors that potentiate cytokine production by human cells through a protease-activated receptor 1- and 2-independent pathway. Infect Immun 78:393–399

    Article  CAS  PubMed  Google Scholar 

  16. Pietrella D, Rachini A, Pandey N, Schild L, Netea M, Bistoni F, Hube B, Vecchiarelli A (2010) The inflammatory response induced by aspartic proteases of Candida albicans is independent of proteolytic activity. Infect Immun 78:4754–4762

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Maza PK, Oliveira P, Toledo MS, Paula DM, Takahashi HK, Straus AH, Suzuki E (2012) Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases. Microbes Infect 14:1077–1085

    Article  CAS  PubMed  Google Scholar 

  18. Hirata I, Cezari MS, Nakaie C, Boschcov P, Ito A, Juliano M, Juliano L (1995) Internally quenched fluorogenic protease substrates: solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs. Lett Pept Sci 1:299–308

    Article  CAS  Google Scholar 

  19. Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan ML, Juliano L, Gauthier F (2008) Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 3:991–1000

    Article  CAS  PubMed  Google Scholar 

  20. Zhang H, Zeng X, He S (2014) Evaluation on potential contributions of protease activated receptors related mediators in allergic inflammation. Mediators Inflamm 2014:829068

    PubMed  PubMed Central  Google Scholar 

  21. Elmariah SB, Reddy VB, Lerner EA (2014) Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist. PLoS ONE 9:e99702

    Article  PubMed  PubMed Central  Google Scholar 

  22. Lourbakos A, Yuan YP, Jenkins AL, Travis J, Andrade-Gordon P, Santulli R, Potempa J, Pike RN (2001) Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. Blood 97:3790–3797

    Article  CAS  PubMed  Google Scholar 

  23. Torres I, Hernandez O, Tamayo D, Muñoz JF, Leitão NP, García AM, Restrepo A, Puccia R, McEwen JG (2013) Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis. PLoS ONE 8:e68434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Barros BCSC, Maza PK, Alcantara C, Suzuki E (2016) Paracoccidioides brasiliensis induces recruitment of α3 and α5 integrins into epithelial cell membrane rafts, leading to cytokine secretion. Microbes Infect 18:68–77

    Article  CAS  PubMed  Google Scholar 

  25. Lerman YV, Lim K, Hyun YM, Falkner KL, Yang H, Pietropaoli AP, Sonnenberg A, Sarangi PP, Kim M (2014) Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrin α3β1-dependent. Blood 124:3515–3523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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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Authors and Affiliations

  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

    Priscila de Oliveira, Bianca Carla Silva Campitelli de Barros, Paloma Korehisa Maza & Erika Suzuki

  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

    Maria Aparecida Juliano & Saara Maria Batista dos Santos

  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

    Aparecida Sadae Tanaka

  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

    Adriana Karaoglanovic Carmona

  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

    Rosana Puccia

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  1. Priscila de Oliveira
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  2. Maria Aparecida Juliano
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  3. Aparecida Sadae Tanaka
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  4. Adriana Karaoglanovic Carmona
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  5. Saara Maria Batista dos Santos
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  6. Bianca Carla Silva Campitelli de Barros
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  7. Paloma Korehisa Maza
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  8. Rosana Puccia
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  9. Erika Suzuki
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Correspondence to Erika Suzuki.

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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

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  • DOI: https://doi.org/10.1007/s00430-016-0490-x

Keywords

  • Protease-activated receptors
  • Epithelial cell
  • Fungus
  • Paracoccidioides
  • Protease
  • Cytokine