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J Med Microbiol 58 (2009), 706-713; DOI: 10.1099/jmm.0.003830-0
© 2009 Society for General Microbiology
ISSN 0022-2615

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Fabiana Cristina Donofrio1, Ana Carolina Alvarez Calil1, Elaine Toscano Miranda1, Ana Marisa Fusco Almeida1, Gil Benard2, Christiane Pienna Soares1, Sarah Nogueira Veloso3, Célia Maria de Almeida Soares3 and Maria José Soares Mendes Giannini1

1 Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil

2 Laboratório de Dermatologia e Imunodeficiências, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil

3 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil

Correspondence
Maria José Soares Mendes Giannini
giannini{at}fcfar.unesp.br

Received May 30, 2008
Accepted January 30, 2009

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Abbreviations: ECM, extracellular matrix.






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