Bacterial-Epithelial Contact Is a Key Determinant of Host Innate Immune Responses to Enteropathogenic and Enteroaggregative Escherichia coli Lindsey A. Edwards1,2*, Mona Bajaj-Elliott2, Nigel J. Klein2, Simon H. Murch3, Alan D. Phillips1 1 Centre for Paediatric Gastroenterology, Royal Free Hospital, London, United Kingdom, 2 Infectious Diseases and Microbiology, Institute of Child health, London, United Kingdom, 3 Division of Metabolic and Vascular Health, Warwick Medical School, Coventry, United Kingdom Abstract Background: Enteropathogenic (EPEC) and Enteroaggregative (EAEC) E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s) remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains. Methodology: Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS) negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence. Results: Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti- inflammatory response. Conclusion: Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbe’s ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis. Citation: Edwards LA, Bajaj-Elliott M, Klein NJ, Murch SH, Phillips AD (2011) Bacterial-Epithelial Contact Is a Key Determinant of Host Innate Immune Responses to Enteropathogenic and Enteroaggregative Escherichia coli. PLoS ONE 6(10): e27030. doi:10.1371/journal.pone.0027030 Editor: Michael B. Fessler, National Institute of Environmental Health Sciences, United States of America Received July 26, 2011; Accepted October 9, 2011; Published October 28, 2011 Copyright: 2011 Edwards et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: LA Edwards was supported by the Crohn’s in childhood Research Appeal (CICRA) charity. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: l.edwards@ich.ucl.ac.uk Introduction Diarrhoeal disease is the second leading cause of infant mortality under the age of 5 worldwide with 1.5 million infant deaths each year, in addition to 1.1 million deaths in adults and infants over the age of 5 [1]. Common amongst the Diarrhoea- genic Escherichia coli strains is the ability to colonise the intestinal mucosa, evade host defences, multiply and cause host damage. One such strain, enteropathogenic E. coli (EPEC), is a human pathogen of the small intestine and is a significant cause of infantile diarrhoea [2]. The pathogenesis of EPEC involves three stages: (I) Initial adherence of bacteria to intestinal epithelial cells (IEC) in a characteristic pattern called ‘‘localised adherence’’ [3]. (II) Modu- lation of signal transduction via a type three secretion system (TTSS). EPEC injects a number of secreted effector proteins (Esp) directly into host cells, which can modulate host inflammation [2,4]. If an inflammatory response ensues, it is due to the host pro- inflammatory response to EPEC outweighing the bacterial TTSS mediated anti-inflammatory response [4]. (III) Intimate adherence via an attaching and effacing (A/E) lesion [5], causing microvillus effacement that results in persistent watery diarrhoea, which ranges from non to weakly inflammatory [6]. Enteroaggregative E. coli (EAEC) is commonly associated with paediatric diarrhoea and malnutrition in developing countries; nevertheless EAEC is emerging as a significant diarrhoeal pathogen in adults, including HIV-positive patients and travellers. EAEC is a leading cause of food-borne outbreaks in the indus- trialized world and has been implicated in the development of post-infectious Irritable Bowel Syndrome [7]. There has been an important recent outbreak of severe haemolytic uraemia syndrome PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e27030