| Abstract: | 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.Microbial adhesion to host tissues is the initial event of most infectious process (39). Interaction with extracellular matrix (ECM) proteins has been correlated with the invasive abilities of different organisms (28, 40). ECM underlines epithelial and endothelial cells and surrounds connective tissues, and its major components are the collagens, laminin, fibronectin, and proteoglycans (52). After adherence, the next step must be to overcome the barriers imposed by epithelial tissues and ECM. The proteolytic activity achieved by subversion of host proteases by pathogens, such as plasmin, has been shown to be important during the process of many types of infections (47, 51).Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis that constitutes a major health problem in South America (44). Clinical manifestations of PCM are related to chronic granulomatous reactions with involvement of the lungs and the reticuloendothelial system, as well as mucocutaneous areas and other organs (22). In the soil, the fungus grows as saprobic mycelium, resulting in the formation of infectious propagules. After penetrating the host, the fungus differentiates into its yeast form, a fundamental step for the successful establishment of the disease (46).Although P. brasiliensis is not traditionally considered a typical intracellular pathogen, independent studies have demonstrated that P. brasiliensis yeast cells have the capacity to adhere and invade host cells (4, 24, 31). P. brasiliensis may actively penetrate the mucocutaneous surface and parasitize epithelial cells, thus evading the host defenses and reaching deeper tissues.Fungal ECM-binding adhesins have been characterized in different models, including P. brasiliensis. Vicentini et al. (49) showed specific binding of the protein gp43 to laminin, which is correlated to the adhesiveness of the fungus in vitro as well as to an enhancement of pathogenic potential. We have been systematically searching for new adhesion proteins in P. brasiliensis that have the potential to play roles in the fungal virulence, and proteins such as P. brasiliensis malate synthase (PbMLS) (34), PbDfg5p (defective for filamentous growth protein) (10), triosephosphate isomerase (PbTPI) (41), and glyceraldehyde-3-phosphate dehydrogenase (PbGAPDH) (4) were found to associate with ECM components. In particular, enolase from P. brasiliensis (PbEno) is a fibronectin-binding protein, as characterized by affinity ligand assays (17).The importance of plasminogen in infectious diseases is supported by the fact that many pathogens manifest the ability to bind plaminogen (47, 13). Plasminogen is a single-chain glycoprotein with a molecular mass of 92 kDa. Its protein structure comprises an N-terminal preactivation peptide, five consecutive disulfide-bonded triple-loop kringle domains, and a serine-protease domain containing the catalytic triad (48). The kringle domains of plasminogen mediate its attachment to cells surfaces by binding proteins with accessible carboxyl-terminal or internal lysine residues. The plasminogen system displays a unique role in the host defense by dissolving fibrin clots and serving as an essential component to maintain homeostasis (43). Activation of the fibrinolytic system is dependent on the conversion of plasminogen to the serine-protease plasmin by the physiological activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) (9). Plasmin is involved in fibrinolysis homeostasis and degradation of the extracellular matrix and basement membrane. The mammalian plasminogen-plasmin proteolytic system plays a crucial role in extracellular matrix degradation which is exploited by invasive pathogens, including fungi (25, 47). Microbe-derived plasminogen conversion to plasmin may promote dissemination of the pathogen within the host (1).Among several proteins, enolase has been found to play a major role in microbial recruitment of plasminogen (32). By serving as a key surface receptor for plasminogen recruitment, enolase has been shown to function as mediator of microbial virulence (6, 15). The potential of P. brasiliensis to recruit human plasminogen for invasion and virulence has not been studied until now. In this study, we demonstrated for the first time that P. brasiliensis is capable of recruiting plasminogen and activating the plasminogen fribrinolytic system in a process, at least in part, mediated by the cell wall-localized enolase. Furthermore, recombinant PbEno (rPbEno) promoted an increase in the adhesion/invasion of P. brasiliensis in in vitro models of infection, a process that seems to be associated with the enolase ability of modifying the surface of host cells. These data suggest that PbEno may play a role in mediating the P. brasiliensis recruitment of plasminogen as well as in attachment and internalization of the fungus to host tissues, potentially playing a role in the establishment of PCM. |
