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Infection and Immunity, April 2005, p. 2486-2495, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2486-2495.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Purification and Partial Characterization of a Paracoccidioides brasiliensis Protein with Capacity To Bind to Extracellular Matrix Proteins

Angel González,1* Beatriz L. Gómez,1,2 Soraya Diez,1,2 Orville Hernández,1 Angela Restrepo,1 Andrew J. Hamilton,2,{dagger} and Luz E. Cano1,3,{dagger}

Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas,1 Molecular Microbiology Group, Bacteriology and Clinical Laboratory School, Universidad de Antioquia, Medellín, Colombia,3 Dermatology Department, Guy's and St. Thomas' Hospital, King's College, London University, London, United Kingdom2

Received 28 July 2004/ Returned for modification 2 September 2004/ Accepted 7 December 2004

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, ß-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.

* Corresponding author. Mailing address: Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), Carrera 72 A, No. 78B 141, A. A. 73 78 Medellín, Colombia. Phone: 57-4-441 08 55. Fax: 57-4-441 55 14. E-mail: agonzalezm{at}cib.org.co.

Editor: T. R. Kozel

{dagger} Luz E. Cano and Andrew J. Hamilton share senior authorship on the manuscript.

Infection and Immunity, April 2005, p. 2486-2495, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2486-2495.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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