Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells.

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.

Protein kinase antagonists inhibit invasion of mammalian cells by Fonsecaea pedrosoi.

The phosphorylation process is an important mechanism of cell signalling and regulation. It has been implicated recently in defence strategies against a variety of pathogens that alter host signalling pathways in order to facilitate their invasion and survival within host cells. In this study, the involvement of protein kinases (PKs) has been investigated in attachment and invasion by the pathogenic fungus Fonsecaea pedrosoi within epithelial cells and macrophages. The use of the PK inhibitors staurosporine, genistein and calphostin C prior to infection provided significant information about the role played by PKs in the F. pedrosoi-host cell interaction. All three PK inhibitors could reduce cell invasion by F. pedrosoi significantly. Pre-treatment of macrophages, epithelial cells or conidia with PK inhibitors decreased fungus invasion, and this effect could be overcome by okadaic acid, a phosphatase inhibitor. Immunofluorescence assays showed that tyrosine residues were phosphorylated in the first step of the interaction, while serine residues were phosphorylated in the subsequent step of entry of the parasite into the host cell. These results suggest that both host-cell and conidium PK activities are important in the interaction process, playing a significant role in cell invasion.