RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins

The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.

Dynamic Interactions of a Conserved Enterotoxigenic Escherichia coli Adhesin with Intestinal Mucins Govern Epithelium Engagement and Toxin Delivery

At present, there is no vaccine for enterotoxigenicEscherichia coli(ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design.