Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth

Abstract

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 β-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. The P. brasiliensis dfg5 nucleotide and deduced protein, PbDfg5p, sequences reported in this paper had been submitted to the GenBank database under Accession Nos AY307855 (cDNA) and DQ534495 (genomic).