Strukturelle und funktionelle Charakterisierung der epithelialen Adhäsine aus Candida glabrata

The human pathogenic fungus Candida glabrata is an opportunistic pathogen that preferentially colonizes the mucosa of mammals. C. glabrata is responsible for about 15 % of blood stream infections in humans and causes particularly serious infections in immunocompromised patients. Important virulence factors of C. glabrata are the epithelial adhesins (Epa proteins) that are crucial for the successful colonization of the host. Depending on the strain background, certain fungal strains express up to 23 different members of this protein family. Epa proteins are related to the flocculins in the baker's yeast Saccharomyces cerevisiae and belong to the group of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWP). They have a typical three-part domain structure in which the N-terminal A domain is responsible for mediating the adhesion, followed by the B domain, which acts as a carrier of the A domain and the C-terminal C domain, which cross-links the adhesin covalently to the cell wall. Previous studies on some members of the Epa family have shown that these adhesins are lectins that identify and specifically bind certain glycans on the surface of epithelial cells. To investigate the structure and function of the entire Epa protein family in detail, all 17 EpaA domains of C. glabrata strain CBS138 were heterologously produced and purified in this work. Subsequently, the ligand binding specificity patterns of all 17 EpaA domains were determined by glycan array screening, which revealed that about 2/3 of the Epa proteins prefer galactosides as binding partners. Moreover, the exact binding strengths of certain EpaA domains to selected glycan ligands were determined, which were in a micromolar range. Furthermore the protein structure of Epa6A and Epa9A with various ligands has been resolved. Thereby it was possible to identify both conserved and variable amino acid residues that are important for the binding affinity and specificity, and which were then investigated in more detail by means of targeted mutagenesis. In order to systematically investigate the function of the EpaA domains in vivo and to compare them with the biochemical results, 15 of the 17 EpaA domains were analyzed in a heterologous expression system, in which each one of the proteins were targeted to the cell surface of S. cerevisiae, so their ability to mediate adhesion to human epithelial cells and abiotic surfaces could be determined. In summary this work gives a detailed insight into the structure and function of a complete multi- protein family. Thereby the precise characterization of ligand binding sites is of particular interest, since these are critical for the pathogenicity of C. glabrata and are potential targets for future development of urgently needed, new antifungal agents.