The Role of Fibronectin in the Adherence and Inflammatory Response Induced by Enteroaggregative Escherichia coli on Epithelial Cells

Dominique Yáñez, Mariana Izquierdo, Fernando Ruiz-Perez, James P. Nataro, Jorge A. Girón, Roberto M. Vidal, Mauricio J. Farfan

Enteroaggregative Escherichia coli (EAEC) infections are still one of the most important etiologic pathogens of diarrhea in children worldwide. EAEC pathogenesis comprises three stages: adherence and colonization, production of toxins, and diarrhea followed by inflammation. Previous studies have demonstrated that EAEC strains have the ability to bind to fibronectin (FN); however, the role this extracellular matrix protein plays in the inflammatory response induced by EAEC remains unknown. In this study, we postulated that FN-mediated adherence of EAEC strains to epithelial cells increases the expression of pro-inflammatory genes. To verify this hypothesis, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with EAEC reference strain 042. We quantified IL-8 secretion and the relative expression of a set of genes regulated by the NF-κB pathway. Although FN increased EAEC adherence, no changes in IL-8 protein secretion or IL8 gene expression were observed. Similar observations were found in HEp-2 cells transfected with FN-siRNA and infected with EAEC. To evaluate the involvement of AAF/II fimbriae, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with an EAEC 042aafA mutant strain transformed with a plasmid harboring the native aafA gene with a site-directed mutation in Lys72 residue (K72A and K72R strains). No changes in IL-8 secretion were observed. Finally, SEM immunogold assay of cells incubated with FN and infected with EAEC revealed that AAF fimbriae can bind to cells either directly or mediated by FN. Our data suggests that FN participates in AAF/II fimbriae-mediated adherence of EAEC to epithelial cells, but not in the inflammatory response of cells infected by this pathogen.