Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells Atul Goyal a , Monica Konar b , Akanksha Setia b , Anil Narang a , Sujata Ghosh b, ⁎ a Department of Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India b Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India abstract article info Article history: Received 19 September 2009 Received in revised form 10 March 2010 Accepted 12 March 2010 Available online 18 March 2010 Keywords: EAEC Adhesin Signaling MAPK STAT-3 IL-8 Background: Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407). Methods: INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgG AD )/D-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA. Results: The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgG AD /D-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490). Conclusions: We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2. General significance: Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection. © 2010 Elsevier B.V. All rights reserved. 1. Introduction EAEC is an important etiological agent of acute and persistent diarrhea in both developing and industrialized countries [1,2]. In developing countries, infectious diarrhea is frequently disabling and contributes substantially to malnutrition, resulting in high morbidity and mortality. A great deal has been learned about the pathogenesis of EAEC infections in the past decade. Nevertheless, our understanding of the pathogenesis of this organism is still suboptimal. Aggregative adherence (AA) to HEp-2 cells is the defining characteristic of EAEC [3]. Adhesin mediated AA of these organisms to intestinal mucosa marks a crucial early event in their interaction with the host resulting in colonization and infection [4,5]. Earlier studies demonstrated that in addition to have an essential role in adherence of the enteric bacteria to intestinal mucosa, the adhesins of these organisms also play an important role in induction of host cell signal transduction during subsequent stages of the infection [6]. Published experimental evidence of our group have revealed the identification and characterization of a novel galactose specific fimbrial adhesin from EAEC-T7 [7]. This adhesin was found to possess a specific role in the AA of this strain to INT-407 cells [8]. Further, the antibody against this adhesin recognized it from crude fimbrial preparations of 9 out of 10 clinical isolates of EAEC strains [9]. Thus, the conserved nature of this adhesin makes it potentially useful to study the molecular mechanisms involved in the pathogenesis of the disease caused by EAEC. Although the mechanism by which EAEC cause disease remains elusive, part of the symptomatology suffered by infected hosts reflects the subsequent host inflammatory responses to infection. Much of this pathology has been linked to inflammatory responses by the infected epithelium, including the production of IL-8 [10,11]. Since the discovery of EAEC-flagellin induced IL-8 release from intestinal epithelial cells [12], other adherence factors have also been assessed for their proinflammatory role [4,13]. Recently, we have reported the Biochimica et Biophysica Acta 1800 (2010) 574–579 ⁎ Corresponding author. Tel.: + 91 172 2755236; fax: + 91 172 2744401. E-mail address: suassi1@gmail.com (S. Ghosh). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.009 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen