Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein.
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.
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Fabiana Cristina
FC
Donofrio
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Ana Carolina Alvarez
ACA
Calil
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Elaine Toscano
ET
Miranda
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Ana Marisa Fusco
AMF
Almeida
Laboratório de Dermatologia e Imunodeficiências, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
Gil
G
Benard
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Christiane Pienna
CP
Soares
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
Sarah Nogueira
SN
Veloso
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
Célia Maria de Almeida
CMA
Soares
Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Maria José Soares
MJS
Mendes Giannini
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D004847:Epithelial Cells / Q000382:microbiology; D005353:Fibronectins / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008168:Lung / Q000166:cytology / Q000382:microbiology; D008297:Male; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000201:enzymology* / Q000472:pathogenicity* / Q000502:physiology; D010229:Paracoccidioidomycosis / Q000382:microbiology*; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D005353:Fibronectins; D005656:Fungal Proteins; D010751:Phosphopyruvate Hydratase