Structural and functional studies of mucin-interacting adhesion domains from Candida glabrata and Helicobacter pylori

Epithelial adhesins from Candida glabrata Epithelial adhesins (Epa) are crucial proteins in the colonization, pathogenesis and virulence of Candida glabrata. These adhesins have a similar modular structure to Saccharomyces cerevisiae flocculins, with an N-terminal adhesive A domain, a central neck-like B domain, and an anchorage C-terminal C domain. A hallmark of many fungal adhesins is the presence of a calcium binding PA14 domain within the A domain. The PA14 domain is responsible for carbohydrate binding in a calcium dependent manner, which allows for the classification of these proteins as C-type lectins[1]. In this study, it was possible to elucidate the crystal structures of Epa1A and three variants at resolutions from 1.4 to 2.0 Å. The latter were meant to emulate the specificities of Epa2, Epa3 and Epa6 adhesive domains. The results yielded a profound knowledge of the binding pocket of Epa1A and the mechanisms through which specificity is controlled in the Epa A domain. Especially surprising was the fact that, even though the proteins were crystallized in the presence of lactose, the protein co-crystals never showed the aforementioned sugar. Instead, a galactoseβ1-3glucose disaccharide unit could be modeled into the electron density. The disaccharide is commonly found on cell surfaces and milk derivates, from which the employed lactose was obtained[2]. Epa1A , Epa1→2A, Epa1→3A and Epa1→6A were also functionally characterized by semi- quantitative, high-throughput methodologies. In collaboration with the consortium for functional glycomics, fluorescently labeled proteins were set in contact with large-scale glycan arrays. The results showed a marked preference for galactoseβ1-3 terminal oligosaccharides in the case of Epa1A. For the other proteins, varying degrees of promiscuity were noted. Epa1→6A presented a very similar binding profile to the one presented by Zupancic et. al. in 2008 for Epa6A, demonstrating the validity of the method. Epa1→2A and Epa1→3A were much less active, and presented a preference for sulfated glycans, along with terminal galactose. Fluorescence titrations showed for Epa1A a ~20 time stronger affinity for the T antigen (galactoseβ1-3N-acetyl- galactosamine) than for the milk-derived lactose, showing how marked the adhesin preference for β1-3 linkages is, as compared to β1-4 glycosidic bonds. Adhesins of Helicobacter pylori The adhesins of H. pylori have been shown to be critical for the colonization and immune recognition of the bacterium during gastric invasion and disease development[3]. BabA and SabA figure prominently, as the former is the primary adhesin during early stage colonization, while the latter binds strongly to inflamed tissue[4]. Both of them are autotransporters, with a C-terminal, membrane bound translocation unit and an N-terminal passenger domain which contains the adhesive portion of the protein[5]. Pure, soluble passenger domains of BabA and SabA were successfully overproduced by recombinant expression in Escherichia coli. BabA could be functionally characterized by the same method as the Epa proteins. The results showed that BabA activity was strongly pH dependent, with a ~100 times stronger activity at pH 5.8 than at pH 2.5. This behavior could be further characterized through circular dichroism spectroscopy and size exclusion chromatography, which showed that BabA is in a reversible molten globule-like, aggregation-prone and relaxed conformation at pH 2.5. At pH 5.8, on the other hand, the protein is in a much more compact, defined conformation with a strong tendency to precipitate. H. pylori has been shown to present many pH dependent virulence factors, like the urea transporter, but up to now no direct biochemical data had been presented supporting pH dependent conformational changes in its adhesins