Abstract
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.
Highlights
Enteroaggregative Escherichia coli (EAEC) are a major cause of acute diarrhea in developing and industrialized regions (Huang et al, 2006)
Using 7.5 μg/ml of FN, we evaluated the amount of IL-8 secreted to the media by HEp-2 and HT-29 cells 3 h post-infection with EAEC
One of the main questions regarding the ability of pathogens to adhere to FN is the biological significance of this binding in the context of disease establishment. It has been recognized for some time that FN plays a role in EAEC 042’s adherence to epithelial cells through interaction with AAF/II fimbriae, the major EAEC virulence factor known to be involved in both adherence and inflammation (Farfan et al, 2008; Konar et al, 2012; Izquierdo et al, 2014a)
Summary
Enteroaggregative Escherichia coli (EAEC) are a major cause of acute diarrhea in developing and industrialized regions (Huang et al, 2006) Several outbreaks, such as the 2011 outbreak caused by a Shiga toxin-producing EAEC in Germany, have increased interest in investigating the molecular mechanisms involved in the interaction between this pathogen and the intestinal epithelia (Rasko et al, 2011). Fibronectin (FN) was the first ECM protein shown to act as a cell receptor for bacterial pathogens (Kuusela, 1978). This glycoprotein contains multiple domains that can bind to several ligands, such as fibrin, heparin, syndecan, collagens, gelatin, and integrins (Pankov and Yamada, 2002). Over 100 bacterial FN binding proteins have been identified, and several studies have shown that adhesion to FN would contribute to the successful colonization of bacteria on the epithelial surface, by acting as a “molecular bridge” connecting the bacteria with the host cell surface (Henderson et al, 2011; Izquierdo et al, 2014a)
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