Mycopathologia

, Volume 179, Issue 3–4, pp 231–242 | Cite as

1,10-Phenanthroline Inhibits the Metallopeptidase Secreted by Phialophora verrucosa and Modulates its Growth, Morphology and Differentiation

  • Marcela Queiroz Granato
  • Priscila de Araújo Massapust
  • Sonia Rozental
  • Celuta Sales Alviano
  • André Luis Souza dos Santos
  • Lucimar Ferreira Kneipp
Article

Abstract

Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.

Keywords

Phialophora verrucosa Chromoblastomycosis Metallopeptidase 1,10-Phenanthroline Growth Differentiation 

References

  1. 1.
    Rippon JW. Chromoblastomycosis. In: Medical mycology: the pathogenic fungi and the pathogenic actinomycetes. WB Saunders: Philadelphia; 1988. p. 276–96.Google Scholar
  2. 2.
    Queiroz-Telles F, Esterre P, Perez-Blanco M. Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol. 2009;47:3–15.CrossRefPubMedGoogle Scholar
  3. 3.
    Santos ALS, Palmeira VF, Rozental S, Kneipp LF, Nimrichter L, Alviano DS, Rodrigues ML, Alviano CS. Biology and pathogenesis of Fonsecaea pedrosoi, the major etiologic agent of chromoblastomycosis. FEMS Microbiol Rev. 2007;31:570–91.CrossRefPubMedGoogle Scholar
  4. 4.
    Ameen M. Managing chromoblastomycosis. Trop Doct. 2010;40:65–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Tong Z, Chen SC, Chen L, Dong B, Li R, Hu Z, Jiang P, Li D, Duan Y. Generalized subcutaneous phaeohyphomycosis caused by Phialophora verrucosa: report of a case and review of literature. Mycopathologia. 2013;175:301–6.CrossRefPubMedGoogle Scholar
  6. 6.
    Turiansky GW, Benson PM, Sperling LC, Sperling LC, Sau P, Salkin IF, McGinnis MR, James WD. Phialophora verrucosa: a new cause of mycetoma. J Am Acad Dermatol. 1995;32:311–5.CrossRefPubMedGoogle Scholar
  7. 7.
    Lundstrom TS, Fairfaz MR, Dugan MC, Vazquez JA, Chandrasekar PH, Abella E, Kasten-Sportes C. Case report Phialophora verrucosa infection in a BMT patient. Bone Marrow Transplant. 1997;20:789–91.CrossRefPubMedGoogle Scholar
  8. 8.
    Campos-Herrero MI, Tandón L, Horcajada I, Medina-Rivero F. Endophthalmitis caused by Phialophora verrucosa: a case report and literature review of Phialophora ocular infections. Enferm Infecc Microbiol Clin. 2012;30:163–5.CrossRefPubMedGoogle Scholar
  9. 9.
    Braga-Silva LA, Santos ALS. Aspartic protease inhibitors as potential anti-Candida albicans drugs: impacts on fungal biology, virulence and pathogenesis. Curr Med Chem. 2011;18:2401–19.CrossRefPubMedGoogle Scholar
  10. 10.
    Monod M, Capoccia S, Léchenne B. Secreted proteases from pathogenic fungi. Int J Med Microbiol. 2002;292:405–19.CrossRefPubMedGoogle Scholar
  11. 11.
    Naglik JR, Challacombr SJ, Hube B. Candida albicans aspartyl proteinase in virulence and pathogenesis. Microbiol Mol Biol Rev. 2003;2003(67):400–28.CrossRefGoogle Scholar
  12. 12.
    Santos ALS. Protease expression by microorganisms and its relevance to crucial physiological/pathological events. World J Biol Chem. 2011;26:48–58.CrossRefGoogle Scholar
  13. 13.
    Silva BA, Santos ALS, Barreto-Bergter E, Pinto MR. Extracellular peptidase in the fungal pathogen Pseudallescheria boydii. Curr Microbiol. 2006;53:18–22.CrossRefPubMedGoogle Scholar
  14. 14.
    Palmeira VF, Kneipp LF, Alviano CS, Santos ALS. The major chromoblastomycosis fungal pathogen, Fonsecaea pedrosoi, extracellularly releases proteolytic enzymes whose expression is modulated by culture medium composition: implications on the fungal development and cleavage of key’s host structures. FEMS Immunol Med Microbiol. 2006;46:21–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Palmeira VF, Kneipp LF, Alviano CS, Santos ALS. Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors. Res Microbiol. 2006;157:819–26.CrossRefPubMedGoogle Scholar
  16. 16.
    Palmeira VF, Kneipp LF, Rozental S, Alviano CS, Santos ALS. Beneficial effects of HIV peptidase inhibitors on Fonsecaea pedrosoi: promising compounds to arrest key fungal biological processes and virulence. PLoS One. 2008;3:3382.CrossRefGoogle Scholar
  17. 17.
    Buroker-Kilgore M, Wang KKA. Coomassie brilliant blue G-250-based colorimetric assay for measuring activity of calpain and other proteases. Anal Biochem. 1993;208:387–92.CrossRefPubMedGoogle Scholar
  18. 18.
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.CrossRefPubMedGoogle Scholar
  19. 19.
    Blum H, Beier H, Gross HJ. Improved silver staining of plant proteins, RNA and DNA polyacrylamide gels. Eletrophoresis. 1987;8:93–9.CrossRefGoogle Scholar
  20. 20.
    CLSI. Clinical Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: second edition (M38-A2). Wayne: CLSI; 2008.Google Scholar
  21. 21.
    Cruz MCS, Santos PO, Barbosa AM Jr, Melo DLFM, Alviano CS, Antoniolli AR, Alviano DS, Trindade RC. Antifungal activity of Brazilian medicinal plants involved in popular treatment of mycoses. J Ethnopharmacol. 2007;111:409–12.CrossRefPubMedGoogle Scholar
  22. 22.
    Vitale RG, Perez-Blanco M, De Hoog GS. In vitro activity of antifungal drugs against Cladophialophora species associated with human chromoblastomycosis. Med Mycol. 2009;47:35–40.CrossRefPubMedGoogle Scholar
  23. 23.
    Pfaller MA, Sheehan DJ, Rex JH. Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clin Microbiol Rev. 2004;17:268–80.CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Guerra CR, Ishida K, Nucci M, Rozental S. Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology. Mem Inst Oswaldo Cruz. 2012;107:582–90.CrossRefPubMedGoogle Scholar
  25. 25.
    Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012;484:186–94.CrossRefPubMedGoogle Scholar
  26. 26.
    Revankar SG, Sutton DA. Melanized fungi in human disease. Clin Microbiol Rev. 2010;23:884–928. Erratum in: Clin Microbiol Rev. 2012;25:720.Google Scholar
  27. 27.
    Ameen M. Managing mycetomas. Trop Doct. 2009;39:66–8.CrossRefPubMedGoogle Scholar
  28. 28.
    Severo CB, Oliveira FM, Pilar EF, Severo LC. Phaeohyphomycosis: a clinical–epidemiological and diagnostic study of eighteen cases in Rio Grande do Sul, Brazil. Mem Inst Oswaldo Cruz. 2012;107:854–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Kneipp LF, Magalhães AS, Abi-Chacra EA, Souza LO, Alviano CS, Santos AL, Meyer-Fernandes JR. Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion. Med Mycol. 2012;50:570–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Liu H, Kauffman S, Becker JM, Szaniszlo PJ. Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection. Eukaryot Cell. 2004;3:40–51.CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Holz RC, Bzymek KP, Swierczek SI. Co-catalytic metallopeptidases as pharmaceutical targets. Curr Opin Chem Biol. 2003;7:197–206.CrossRefPubMedGoogle Scholar
  32. 32.
    Silva BA, Pinto MR, Soares RMA, Barreto-Bergter E, Santos ALS. Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds. Res Microbiol. 2006;157:425–32.CrossRefPubMedGoogle Scholar
  33. 33.
    Silva BA, Souza-Gonçalves AL, Pinto MR, Barreto-Bergter E, Santos ALS. Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum. Mycoses. 2009;54:105–12.CrossRefGoogle Scholar
  34. 34.
    Gravi ET, Paschoalin T, Dias BR, Moreira DF, Belizario JE, Oliveira V, Carmona AK, Juliano MA, Travassos LR, Rodrigues EG. Identification of a metallopeptidase with TOP-like activity in Paracoccidioides brasiliensis, with increased expression in a virulent strain. Med Mycol. 2012;50:81–90.CrossRefPubMedGoogle Scholar
  35. 35.
    McCann M, Kellett AM, Kavanagh K, Devereux M, Santos ALS. Deciphering the antimicrobial activity of phenanthroline chelators. Curr Med Chem. 2012;19:2703–14.CrossRefPubMedGoogle Scholar
  36. 36.
    Sammes PG, Yahioglu GY. 1,10-Phenanthroline: a versatile ligand. Chem Soc Rev. 1994;23:327–34.CrossRefGoogle Scholar
  37. 37.
    Hooper NM. Families of zinc metalloproteases. FEBS Lett. 1994;354:1–6.CrossRefPubMedGoogle Scholar
  38. 38.
    Valle BL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry. 1990;29:5647–59.CrossRefGoogle Scholar
  39. 39.
    Brul S, Stratford M, Van der Vaart JM, Dielbandhoesing SK, Steels H, Klis FM, Verrips CT. The antifungal action of 1,10-phenanthroline and EDTA is mediated through zinc chelation and involves cell wall construction. Food Technol Biotech. 1997;35:267–74.Google Scholar
  40. 40.
    Singh B, Fleury C, Jalalvand F, Riesbeck K. Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host. FEMS Microbiol Rev. 2012;36:1122–80.CrossRefPubMedGoogle Scholar
  41. 41.
    Yike I. Fungal proteases and their pathophysiological effects. Mycopathology. 2011;171:299–323.CrossRefGoogle Scholar
  42. 42.
    Santos ALS, Sodré CL, Valle RS, Silva BA, Abi-Chacra EA, Silva LV, Souza-Goncalves AL, Sangenito LS, Goncalves DS, Souza LO, Palmeira VF, d’Avila-Levy CM, Kneipp LF, Kellett A, McCann M, Branquinha MH. Antimicrobial action of chelating agents: repercussions on the microorganism development, virulence and pathogenesis. Curr Med Chem. 2012;19:2715–37.CrossRefPubMedGoogle Scholar
  43. 43.
    McCann M, Geraghty M, Devereux M, Devereux M, O’Shea D, Mason J, O’Sullivan L. Insights into the mode of action of the anti-Candida activity of 1,10-phenanthroline and its metal chelates. Met Based Drugs. 2000;7:185–93.CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B. Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect Ther. 2012;10:85–93.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Marcela Queiroz Granato
    • 1
  • Priscila de Araújo Massapust
    • 1
  • Sonia Rozental
    • 2
  • Celuta Sales Alviano
    • 3
  • André Luis Souza dos Santos
    • 4
    • 5
  • Lucimar Ferreira Kneipp
    • 1
  1. 1.Laboratório de Taxonomia, Bioquímica e Bioprospecção de Fungos, Instituto Oswaldo CruzFundação Oswaldo CruzRio de JaneiroBrazil
  2. 2.Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de Janeiro (UFRJ)Rio de JaneiroBrazil
  3. 3.Laboratório de Superfície de Microrganismos, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de GóesUniversidade Federal do Rio de Janeiro (UFRJ)Rio de JaneiroBrazil
  4. 4.Laboratório de Investigação de Peptidases, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de GóesUniversidade Federal do Rio de Janeiro (UFRJ)Rio de JaneiroBrazil
  5. 5.Programa de Pós-Graduação em Bioquímica, Instituto de QuímicaUniversidade Federal do Rio de Janeiro (UFRJ)Rio de JaneiroBrazil

Personalised recommendations