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

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 twodimensional 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.

A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

“…Although some P. brasiliensis molecules that may participate in adhesion to host tissues have been identified (e.g., malate synthase, enolase, thriose phosphate isomerase, an adaptinlike protein, and glyceraldehyde-3-phosphate dehydrogenase, among others), the exact role of these proteins remains uncharacterized due to a lack of gene-based evidence (3,4,8,10,26).…”

Decreased expression of 14-3-3 inParacoccidioides brasiliensisconfirms its involvement in fungal pathogenesis

“…The membranes were blocked with a solution containing PBS, 5% skim milk and 1% bovine serum albumin (BSA) for 4 h at room temperature. After three washes with PBS, the membranes were incubated with the monoclonal anti-14-3-3 (Imuny Ò , diluted 1:500) antibody overnight, and this assay was also performed with polyclonal anti-enolase 21 as an internal control. The anti-IgG secondary antibody conjugated with peroxidase (Sigma Ò ) diluted 1:1000 was added after further washing, and the 14-3-3 and enolase proteins were visualized by adding 3,3 0 diaminobenzidine and hydrogen peroxide as a substrate.…”

“…The ability to adhere is a biological phenomenon that is widely distributed and shared by many pathogens, enabling the colonization of their respective habitats. 15,16 Some adhesins have been described for P. brasiliensis, such as gp43, 17,18 glyceraldehyde-3-phosphate dehydrogenase, 19 triose phosphate isomerase, 20 enolase, 21,22 a 32-Kda hydrolase, 23,24 malate synthase, 25 isocitrate lyase 26 and the 14-3-3 protein (30 KDa) 27,28 ; this last adhesin is the focus of the present study.…”

Update on Fungal Disease: From Establish Infection to Clinical Manifestation

“…In this context, many adhesions have been described for Paracoccidioides spp. [204][205][206][207][208][209][210][211][212][213][214][215][216][217]. Important virulence factors for these fungi include thermal dimorphism which allows the pathogen to adapt/survive inside the host, and the capacity to produce biofilm in vitro, expression of phospholipase and melanin production [218][219][220][221][222][223].…”