RESUMO
The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p (defective for filamentous growth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a beta-glucan linked protein that undergoes N-glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , Ácido 2-Metil-4-clorofenoxiacético , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , DNA Fúngico/genética , Dicamba , Combinação de Medicamentos , Fluorenos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genoma Fúngico , Humanos , Dados de Sequência Molecular , Paracoccidioides/genética , Paracoccidioides/ultraestrutura , Paracoccidioidomicose/sangue , Paracoccidioidomicose/imunologia , Ligação ProteicaRESUMO
A cDNA encoding a chaperone ClpA homologue of Paracoccidioides brasiliensis was isolated and characterized. The ClpA belongs to a group of ClpATPAses proteins, which are highly conserved, and include several heat inducible molecular chaperones. In this study, a 2879 bp cDNA designated as Pbclpa was obtained which encodes a predicted protein of 927 amino acids. Characteristic consensus motifs of the ClpATPases family are present. The PbClpA middle region was compared to other related ClpA and ClpB proteins from fungi and bacteria. Comparative analysis demonstrated in the middle region the presence of a heptad repeat sequence, characteristic of ClpBs from prokaryotes and fungi, which are absent in ClpAs from prokaryotes but were present in all described fungal ClpAs. Our comparative analysis reveals that one of the criteria typically used to distinguish the prokaryotic subfamilies ClpA and ClpB, the size of the middle sequence, may not be useful in fungi. Phylogenetic analyses were performed with the complete sequences of ClpAs from fungi and bacteria and with the middle regions of those ClpAs present at NCBI and Pfam databases. Our results indicated that both types of analysis can be useful as a tool in the determination of phylogenetic relationships.