Background

EAEC is an important cause of endemic and epidemic diarrheal disease worldwide. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in immunocompromised adults, travelers and food-borne outbreaks in the industrialized world, such as the large lethal outbreak caused by a Shiga toxin (Stx) type 2a-producing EAEC strain of serotype O104:H4 in Northern Europe in 2011 (Harrington et al., 2006; Rasko et al., 2011). EAEC pathogenesis is determined by the organism’s ability to adhere to intestinal cells, produce enterotoxins and cytotoxins, and ultimately to induce inflammation (Harrington et al., 2006). EAEC adherence to intestinal cells is mediated by AAF fimbrial adhesins (Czeczulin et al., 1997). To date, at least five variants of the AAF fimbriae have been described, all encoded in virulence plasmids ranging from 55 to 65 MDa (Jonsson et al., 2015). The AAF structure is comprised by a positively charged major subunit and a putative minor subunit at the tip of fimbrial structures (Berry et al., 2014).

The prototype EAEC strain 042 that exhibits the AAF/II variant has been shown to produce diarrhea in adult volunteers (Nataro et al., 1995). Clinical and laboratory data suggest that EAEC induces inflammatory enteritis, while studies using polarized T84 monolayers indicates that release of IL-8 is associated to the presence of AAF/II adhesin (Harrington et al., 2005). Although the importance of the adherence of EAEC to intestinal cells has been established, the cell receptors involved in the inflammatory response mediated by AAF fimbriae have not been fully characterized. Several receptors on epithelial cells have been identified for AAF/II including extracellular matrix (ECM) proteins such as fibronectin and laminin, and cytokeratin 8 (Farfan et al., 2008; Izquierdo et al., 2014). However, these receptors are localized on the basolateral side of intestinal cells. Thus, it is unlikely that these proteins play an important role during the initial infection with EAEC. Furthermore, it has been shown that fibronectin does not participate in the inflammatory response mediated by AAF/II (Yanez et al., 2016). We have recently found that EAEC also binds to the signaling Muc1 glycoprotein, and such binding is dependent on the sialylation of the protein (Boll et al., 2017). Mucins (MUC) are large (> 200 kDa) secreted and transmembrane glycoproteins with a high carbohydrate content (50-90% by weight) expressed by a variety of normal and malignant secretory epithelial cells (Corfield et al., 2001). Muc1 is a polymorphic transmembrane mucin-like protein that contains a large extracellular domain consisting of a glycosylated polypeptide made up of 30-100 tandem repeats of a 20-amino acid sequence, a transmembrane domain, and a cytoplasmic tail of 72 amino acids (Nath and Mukherjee, 2014). Muc1 is associated to numerous signaling pathways in malignant and inflammatory processes (Nath and Mukherjee, 2014). Our recent study shows that Muc1 is associated with the inflammatory response mediated by AAF/II. Moreover, EAEC 042 up-regulates epithelial Muc1 expression dependent on the presence of AAF (Boll et al., 2017).

The existence of multiple receptors for EAEC in intestinal cells complicates the identification and characterization of specific receptors in this cell lineage. The use of HEK293 cells, which differ from enterocytes, allows the evaluation of new potential receptors through transfection of these cells with plasmids encoding the candidate receptor. Likewise, binding assays performed with cells in suspension minimizes the binding of EAEC strains to non-abiotic surfaces. This protocol could be used to find out potential receptors for other enteropathogens. Here, we described in detail the method we previously used to visualize the adherence of EAEC to HEK293 cells transfected with a Muc1-encoding plasmid.