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Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications
Published on: October 30, 2013
Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to
Angel González1, Beatriz L Gómez, Soraya Diez
1Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), Carrera 72 A, No. 78B 141, A. A. 73 78 Medellín, Colombia. agonzalezm@cib.org.co
Abstract:
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, beta-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.
Insights
Researchers identified two fungal proteins in Paracoccidioides brasiliensis that bind to extracellular matrix proteins. These proteins are crucial for fungal adherence and may play a role in disease spread.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Microorganisms utilize surface molecules to adhere to host extracellular matrix (ECM) proteins.
- Paracoccidioides brasiliensis conidia exhibit interactions with ECM proteins, suggesting the presence of specific fungal adhesins.
Purpose of the Study:
- To identify and characterize fungal proteins from Paracoccidioides brasiliensis that interact with ECM proteins like fibronectin, fibrinogen, and laminin.
- To investigate the role of these proteins in fungal adherence and potential dissemination.
Main Methods:
- Preparation of various extracts (total homogenates, beta-mercaptoethanol, SDS) from P. brasiliensis mycelial and yeast forms.
- Ligand affinity assays using fibronectin, fibrinogen, and laminin to detect interacting polypeptides.
- Purification and N-terminal amino acid sequencing of a 32-kDa protein, and development of a monoclonal antibody (MAb) for further analysis.
- Immunofluorescence and immunoelectron microscopy to determine protein localization and distribution.
- Inhibition assays using the purified protein and MAb to assess their effect on conidial adherence.
Main Results:
- Two polypeptides of 19 and 32 kDa were detected in P. brasiliensis extracts, interacting with fibronectin, fibrinogen, and laminin.
- The 32-kDa protein exhibited homology with hypothetical proteins from related fungal species.
- A MAb against the 32-kDa protein recognized it in both fungal forms and localized it to cell walls and cytoplasm.
- Both the purified 32-kDa protein and the MAb dose-dependently inhibited conidial adherence to ECM proteins.
Conclusions:
- Paracoccidioides brasiliensis possesses at least two surface polypeptides capable of interacting with multiple ECM proteins.
- These identified proteins likely serve as common receptors for fibronectin, fibrinogen, and laminin.
- The findings highlight the critical role of these adhesins in initial conidial attachment and potential dissemination of paracoccidioidomycosis.
