RESUMEN
Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/microbiología , Paracoccidioides/metabolismo , Paracoccidioidomicosis/microbiología , Secuencia de Aminoácidos , Animales , Antígenos Fúngicos/química , Antígenos Fúngicos/genética , Antígenos Fúngicos/metabolismo , Adhesión Celular/fisiología , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Citometría de Flujo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Paracoccidioides/genética , Paracoccidioidomicosis/metabolismo , Fragmentos de Péptidos/metabolismo , Alineación de Secuencia , Células VeroRESUMEN
Paracoccidioides brasiliensis (Pb) yeast cells can enter mammalian cells and probably manipulate the host cell environment to favor their own growth and survival. We studied the uptake of strain Pb 18 into A549 lung and Vero epithelial cells, with an emphasis on the repercussions in the cytoskeleton and the apoptosis of host cells. Cytoskeleton components of the host cells, such as actin and tubulin, were involved in the P. brasiliensis invasion process. Cytochalasin D and colchicine treatment substantially reduced invasion, indicating the functional participation of microfilaments (MFs) and microtubules (MTs) in this mechanism. Cytokeratin could also play a role in the P. brasiliensis interaction with the host. Gp43 was recognized by anti-actin and anti-cytokeratin antibodies, but not by anti-tubulin. The apoptosis induced by this fungus in infected epithelial cells was demonstrated by various techniques: TUNEL, DNA fragmentation and Bak and Bcl-2 immunocytochemical expression. DNA fragmentation was observed in infected cells but not in uninfected ones, by both TUNEL and gel electrophoresis methods. Moreover, Bcl-2 and Bak did not show any differences until 24 h after infection of cells, suggesting a competitive mechanism that allows persistence of infection. Overexpression of Bak was observed after 48 h, indicating the loss of competition between death and survival signals. In conclusion, the mechanisms of invasion of host cells, persistence within them, and the subsequent induction of apoptosis of such cells may explain the efficient dissemination of P. brasiliensis.