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Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein
Fabiana Cristina Donofrio1, Ana Carolina Alvarez Calil1, Elaine Toscano Miranda1
1Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil.
Abstract:
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.
Insights
Paracoccidioides brasiliensis yeast uses a fibronectin-binding adhesin, identified as enolase, to attach to lung cells. This interaction is crucial for fungal adhesion and can be blocked by specific antibodies.
Area of Science:
- Mycology
- Infectious Diseases
- Cell Biology
Background:
- Paracoccidioides brasiliensis invades host cells, manipulating the environment for survival.
- The fungus interacts with host extracellular matrix proteins like fibronectin.
Purpose of the Study:
- To isolate and characterize a fibronectin-binding protein from P. brasiliensis.
- To compare P. brasiliensis strains (Pb18a and Pb18b) regarding adhesion and invasion.
- To investigate the role of fibronectin-binding proteins in fungal adhesion to pulmonary epithelial cells.
Main Methods:
- Two-dimensional electrophoresis and SDS-PAGE to analyze protein expression.
- Ligand affinity binding assays to identify fibronectin-binding proteins.
- Tryptic digestion and mass spectroscopy for protein characterization.
- Inhibition assays using antibodies against the identified protein.
Main Results:
- Protein expression levels varied between culture media (blood agar vs. Fava-Netto).
- Both Pb18a and Pb18b showed stronger adhesion to A549 cells when cultured on blood agar.
- A 54 kDa fibronectin-binding adhesin, identified as P. brasiliensis enolase, was isolated.
- An antibody against the 54 kDa protein inhibited 80% of fungal adhesion to A549 cells.
Conclusions:
- P. brasiliensis produces a fibronectin-binding adhesin (enolase) regardless of culture medium.
- This adhesin plays a significant role in the fungus's attachment to pulmonary epithelial cells.
- Specific antibodies can inhibit this adhesion process, suggesting a potential therapeutic target.
