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

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, ␤-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.

Pulmonary immune responses induced in BALB/c mice by Paracoccidioides brasiliensis conidia

“…This type of adhesion is mediated by specific fungal cell-surface components called adhesins, which frequently bind ECM proteins [104]. Recently, the presence of these adhesins on the surface of both P. brasiliensis conidia and yeast cells has been demonstrated [100,101,105]. The data described above strongly suggest that ECM proteins, especially fibronectin, fibrinogen, laminin and collagen appear to play a significant role in PCM pathogenesis.…”

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

“…Thus, a combination of these two adhesins significantly decreases the adhesion and invasion indices [40]. P. brasiliensis also shows two cellsurface proteins, of 32 and 19 kDa molecular mass that interact with several ECM proteins, such as laminin, fibronectin, and fibrinogen [41]. More recently, recombinant 3-glyceraldeyde phosphate dehydrogenase (GAPDH) from P. brasiliensis was found to be capable of binding to laminin, collagen I, and fibronectin.…”

Pulmonary Paracoccidioidomycosis: Clinical, Immunological and Histopathological Aspects

“…Interestingly, this increased deposition of ECM proteins in the lungs of infected mice was accompanied by a marked afflux of pro-inflammatory cells (González et al, 2008c). In addition, P. brasiliensis is known to express adhesin-like molecules on its surface that recognize and bind to ECM proteins (Barbosa et al, 2006;González et al, 2005a). More recently, in elegant experiments using antisense technology, the role of a P. brasiliensisadhesin molecule was confirmed when a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, conferred both adherence capacity to pulmonary human epithelial cells and virulence in a mouse model of infection The observations described above may suggest that ECM proteins modulate the pathogenesis of PCM by means of two mechanisms: 1) participation in the migration of proinflammatory cells into the lungs and 2) serving as a binding protein for P. brasiliensis expressing on its surface adhesin-like molecules (receptors).…”

“…In addition, it has been reported that the different fungal morphotypes of P. brasiliensis (conidia, yeast cells and mycelia) exhibit on their surface adhesin-type molecules that allow both binding to several ECM proteins -mainly fibronectin, fibrinogen and laminin -and adherence to epithelial cells (Caro et al, 2008;González et al, 2005aGonzález et al, , 2008aHernández et al, 2010). Activation of pulmonary cells, mainly Ms, took place after fungal interaction, thus initiating the inflammatory process through production of pro-inflammatory cytokines and chemokines, which in turn induced the expression of adhesion molecules on the leukocytes' surface (González et al, 2003(González et al, , 2005b.…”