Identification of key proteins of cultured human intestinal cells involved in interaction with enteroaggregativeEscherichia coli

Abstract

Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is known to be mediated by aggregative adherence fimbriae (AAF). However, the host cell molecules involved in EAEC adherence remain uncharacterized. In the present study, four key membrane glycoproteins of cultured human intestinal epithelial cells (INT-407) were found to be involved in the interaction with the T8 strain of EAEC. Nine membrane proteins of INT-407 cells were found to interact with EAEC-T8, of which four were identified as fibronectin, epidermal growth factor receptor (EGFR), Thrombospondin-1 (TSP1) and glucose-regulated protein (GRP-94). Our findings were validated by Western blot using antibody against each identified protein. The adherence of EAEC-T8 to INT-407 cells was reduced to c. 26, 29, 37 and 76% in the presence of the antibody against GRP-94, EGFR, fibronectin and TSP-1, respectively. These findings were further substantiated by flow cytometry, where the final mean fluorescence intensity value of the INT-407 cells (c. 1075) resulting from adherent-labelled bacteria was found to be reduced to c. 26, 132, 228 and 597 in the presence of antibody against GRP-94, EGFR, fibronectin and TSP-1. We propose that GRP-94, EGFR, fibronectin and TSP-1 are involved in the aggregative adherence of EAEC-T8 to INT-407 cells.

Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli

“…It is also plausible that a natural receptor for AAF adhesins is not fibronectin itself, but another or multiple extracellular protein(s). In addition to fibronectin, other host receptors have been implicated in EAEC adhesion [51] and more recently both laminin and cytokeratin8 (CK8) have been confirmed to interact with the major subunit of AAF/II fimbriae [52]. It is conceivable that AAF have evolved a more generalised electrostatic mechanism for binding several host receptors that have appropriate arrangements of acidic residues, negatively-charged glycosylation sugars and/or phosphorylated site.…”

Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

“…In the search of potential receptors for the EAEC, our group determined that EAEC strain 042 could adhere to ECM proteins laminin, type IV collagen, and fibronectin, a process mediated by the AAF/II fimbriae (9). Soon after, using an AAF/II-producing EAEC, four proteins were identified as potential cell receptors: fibronectin, EGFR, TSP1, and GRP-94 (10). Intestinal cells preincubated with antibodies against these molecules showed reduced but not abolished adherence of EAEC to cells.…”

“…We previously showed binding of AAF/II to extracellular matrix (ECM) proteins, such as fibronectin, laminin, and type IV collagen, which enhanced bacterial adherence to the surface of polarized intestinal monolayers (9). Using a proteomics approach, the epidermal growth factor receptor (EGFR), Thrombospondin-1 (TSP1), glucose-regulated protein (GRP-94), and fibronectin were identified as potential receptors for an AAF/IIproducing strain (10). Although significant progress in the discovery of receptors for EAEC has been made, the blocking of known receptors did not cause full inhibition of bacterial binding, suggesting that other receptors for AAF/II might exist in intestinal cells.…”

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

“…During infection of the gastrointestinal tract, EAEC adherence to intestinal epithelial cells stimulates the release of IL-8 and CCL20, which recruits neutrophils and stimulates inflammatory diarrhea (599,600,642,643). Proteomics studies with the EAEC-T8 strain (644) have shown that it interacts with Gp96, a cell surface protein that has significant homology with Hsp90, a protein which triggers signaling pathways in response to binding by other pathogens and leads to activation of inflammatory/immune responses through NF-B and MAPK (601,644,645). In the same proteomics study (644), it was also found that the EAEC-T8 strain interacts with TSP1, an ECM glycoprotein that is rapidly secreted at high levels in inflamed and damaged tissues and has been implicated as a molecular bridge between Gram-positive pathogens and host tissue cells (603,644).…”

“…Proteomics studies with the EAEC-T8 strain (644) have shown that it interacts with Gp96, a cell surface protein that has significant homology with Hsp90, a protein which triggers signaling pathways in response to binding by other pathogens and leads to activation of inflammatory/immune responses through NF-B and MAPK (601,644,645). In the same proteomics study (644), it was also found that the EAEC-T8 strain interacts with TSP1, an ECM glycoprotein that is rapidly secreted at high levels in inflamed and damaged tissues and has been implicated as a molecular bridge between Gram-positive pathogens and host tissue cells (603,644). Additionally, AAF/II can elicit a basolateral release of IL-8 from polarized monolayers of T84 human colonic epithelial cells (603,646).…”