Functional reprogramming of Candida glabrata epithelial adhesins by exchange of variable structural motifs
The yeast Candida glabrata is part of the human microbiome and usually employs a commensal lifestyle, but this fungus is also able to act as an opportunistic pathogen, causing localized as well as severe systemic infections. For host invasion and dissemination, C. glabrata disposes of a large number...
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|The yeast Candida glabrata is part of the human microbiome and usually employs a commensal lifestyle, but this fungus is also able to act as an opportunistic pathogen, causing localized as well as severe systemic infections. For host invasion and dissemination, C. glabrata disposes of a large number of cell wall attached proteins, the most prominent of which are the epithelial adhesins (Epas). The Epa family encompasses more than 20 members, which act as lectins. All Epa paralogs share the common tripartite architecture of fungal adhesins, composed of an N-terminal A domain (adhesion domain), a central B domain consisting of a variable number of serine- and threonine-rich repeats, and a C-terminal region carrying a glycosylphosphatidylinositol (GPI) anchor for attachment to the cell wall. The lectin function of Epa adhesins is conferred by a combination of conserved and variable structural elements within their A domains. Together, these elements form an inner and outer binding pocket and are thought to control ligand binding affinity and specificity.
In this work, variable structural elements of several Epa paralogs were functionally characterized using structure-based mutational analysis, to precisely elucidate their role in conferring host cell adhesion and ligand binding specificity. For this purpose, an array of chimeric EpaA variants carrying directed exchanges of highly variable regions in the inner and the outer binding pocket were constructed and functionally characterized. In vivo adhesion assays with human epithelial cells revealed that both of these structural elements are involved in host cell binding. Specifically, exchanges within the inner binding pocket resulted in a lower binding strength. In contrast, the exchange of elongated loops in the outer binding pocket for shorter variants showed a significant increase in host cell adhesion, whereas chimeras carrying longer instead of shorter loops exhibited reduced adhesion. Chimeric EpaA variants were further characterized by glycan array analysis and fluorescence titration spectroscopy. These measurements demonstrated that the ligand binding specificity of EpaA domains can in principal be reprogrammed by exchange of structural elements in the inner binding pocket, with albeit limited predictability. In contrast, exchanges of outer binding pocket elements generally did not affect ligand binding patterns. For further structural characterization of elongated loops in the outer binding pocket, soaking experiments were performed using protein crystals and complex glycan structures. Since this approach did not yield structural data, the flexibility of long loops was analyzed by molecular dynamic simulations, in order to test a putative lid functionality. These simulations showed that in the absence of glycan ligands, the elongated loop can principally adopt a stable conformation, but does not cover the binding pocket. In the presence of a tetrameric glycan, however, the reducing end of the ligand was stabilized by direct contact with the loop, indicating a crucial function of this variable structural element in binding complex glycan structures.
In a further part of this thesis, the function of variable amino acid residues within the inner binding pocket was investigated which have been postulated to confer specific binding of sulfated glycans. To test this hypothesis, corresponding residues were functionally characterized by mutational analysis in combination with in vivo adhesion tests to human epithelial cells and in vitro ligand binding studies. Interestingly, no correlation was detected between mutated positions and specific sulfoglycan binding. However, docking simulations with sulfated disaccharide ligands suggest that other steric effects control the precise fitting of spatially demanding sulfate groups into the binding pockets of EpaA domains.
In summary, results obtained in this work support the view, that variation of several structural elements in the inner and outer ligand binding pocket of Epa adhesins is a main driver of their functional diversification and evolution.