Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall ofSporothrix schenckiiinvolved in fungal adherence to dermal extracellular matrix

Abstract

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis and an emerging disease in immunocompromised patients. Adherence to target cells is a prerequisite for fungal dissemination and systemic complications. However, information on the cell surface components involved in this interaction is rather scarce. In this investigation, the extraction of isolated cell walls from the yeast phase of S. schenckii with SDS and separation of proteins by SDS-PAGE led to the identification of a periodic acid-Schiff (PAS)-reacting 70 kDa glycoprotein (Gp70) that was purified by elution from electrophoresis gels. The purified glycopeptide exhibited a pI of 4.1 and about 5.7% of its molecular mass was contributed by N-linked glycans with no evidence for O-linked oligosaccharides. Confocal analysis of immunofluorescence assays with polyclonal antibodies directed towards Gp70 revealed a rather uniform distribution of the antigen at the cell surface with no distinguishable differences among three different isolates. Localization of Gp70 at the cell surface was confirmed by immunogold staining. Gp70 seems specific for S. schenckii as no immunoreaction was observed in SDS-extracts from other pathogenic and non-pathogenic fungi. Yeast cells of the fungus abundantly adhered to the dermis of mouse tails and the anti-Gp70 serum reduced this process in a concentration-dependent manner. Results are discussed in terms of the potential role of Gp70 in the host-pathogen interaction.

2D-immunoblotting analysis of Sporothrix schenckii cell wall

“…Scott and Muchmore (1989) demonstrated that yeast-like cells of S. schenckii contain two proteins of 40 and 70-kDa as the major antigens recognized by sera obtained from sporotrichosis patients; also, it was demonstrated that sera from S. schenckiiinfected mice reacted mainly with a 70-kDa antigen (Nascimento & Almeida 2005). In addition, we have purified and characterized a 70-kDa glycoprotein (Gp70) from the cell wall of S. schenckii yeast-like cells that exhibited adhesion properties (Ruiz-Baca et al 2009). Blotting with a polyclonal anti-Gp70 serum revealed a single spot with a pI of 4.1, which suggested that Gp70 is a S. schenckii-specific protein as it was not immunodetected in other pathogenic and non-pathogenic fungi Here, five antigens of 48, 55, 66, 67 and 70 kDa were immunodetected in the mycelial phase of S. schenckii ATCC 58251, whereas only two antigens of 60 and 70 kDa were identified in yeast cells.…”

“…Glycoprotein staining -Proteins transferred to nitrocellulose membranes after 2D-PAGE were stained with periodic acid-Schiff (PAS) reagent as described previously (Ruiz-Baca et al 2009). …”

“…Thus far, a peptide-rhamnomannan and peptide-rhamnogalactan have been isolated and characterized from wall preparations of yeast-like cells (Lloyd & Bitoon 1971, Nakamura 1976, Lopes-Bezerra et al 2006). In addition, there are some reports dealing with the role of a cell wall glycoprotein of 70 kDa in adhesion of S. schenckii to host tissues and extracellular matrix components as well as fungal pathogenesis (Nascimento et al 2008, Ruiz-Baca et al 2009, Teixeira et al 2009). …”

“…Briefly, 1 mL aliquots containing 1 x 10 8 formaldehydekilled yeast-like cells were inoculated into the marginal ear vein of the animal three times per week for two weeks and one week after the final injection the rabbit was bled. The blood serum was saved and stored at -70ºC in aliquots of 2 mL containing 0.01% (w/v) sodium azide.Immunoblotting assays -Immunodetection was carried out as previously described (Mora-Montes et al 2008), except that a primary polyclonal anti-S. schenckii antibody diluted 1:500 in phosphate buffered salineTween 20 (0.05%) was used.Glycoprotein staining -Proteins transferred to nitrocellulose membranes after 2D-PAGE were stained with periodic acid-Schiff (PAS) reagent as described previously (Ruiz-Baca et al 2009). …”

“…To isolate cell walls, the cell homogenate was centrifuged for 15 min at 1,300 g and 4ºC and the pellet was saved and washed five times with lysis buffer to eliminate any intracellular component associated with the walls during the cell disruption process. Cell wall proteins were extracted with hot 2% sodium dodecyl sulfate (SDS) as previously described (Ruiz-Baca et al 2009). Proteins were precipitated with 70% (v/v) ethanol at -20ºC for 5 h and kept at -70ºC until use.…”