Identification of proteins involved in the adhesionof Candida species to different medical devices.

Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.

Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

Entamoeba invadens, encystation process and enolase.

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.