with unusually high mortality in German adults caused by a Shiga- toxin-producing EAEC (O104:H4) [8]. EAEC is defined by its distinctive ‘‘stacked-brick’’ aggregative adherence pattern [9]. The pathogenesis of EAEC involves three stages: (I) Adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF) and adhesins. (II) Increased production of mucus that encrusts EAEC on the surface of enterocytes. (III) Release of toxins and elicitation of an inflammatory response and intestinal secretion. The AAF adhesin (AAF/II) also induces loss of epithelial integrity and delocalization of tight junction proteins, which may facilitate bacterial translocation to the submucosa [10]. Clinically, EAEC infection produces watery diarrhoea, occasionally with blood and mucus, and patients typically manifest intestinal inflammation, with production of pro-inflammatory cytokines such as IL-8 [7]. EPEC and EAEC possess several microbial-associated molec- ular patterns (MAMPs) recognized by host pattern recognition receptor (PRR) families, such as Toll-like receptors (TLR). Despite extensive studies the structures that specifically induce the IEC inflammatory response have not been unequivocally identified. Recent studies have highlighted the role of EPEC and EAEC flagellin monomer (FliC) alone in mediating IEC NF-kB and p38 MAPK activation leading to IL-8 production and gastroenteritis [6,11]. In contrast to the well-established effect of FliC, the effect of bacterial adherence to IEC innate response(s) remains unclear. As past findings suggest IL-8 production requires the TTSS and intimate adherence in the case of EPEC [12] and the bacterial aggregative adherence plasmid (pAA) in the case of EAEC [13]. In the present study the aim was to characterise and identify, which bacterial motifs contribute to the production of IL-8 and CCL20. We tested the hypothesis that bacterial adherence contributes to IEC antimicrobial innate immunity during EPEC/EAEC infec- tion. For this purpose an array of isogenic mutants were employed in co-culture studies; our data suggests that ‘epithelial contact’ is indeed a key determinant defining IEC-pathogen crosstalk. Results Bacterial morphology by negative staining transmission electron microscopy (TEM) To gain a greater insight into bacterial-host interactions we determined the presence of microbial structural components known to interact with host PPRs, using negative staining followed by TEM. The E69 WT was found to express a monotrichous flagellum as well as outer membrane vesicles (OMV) (Figure 1a). OMVs are spherical bi-layer vesicles, which are constantly discharged from the bacterial surface during normal growth and can be considered virulence factors [14]. The rough LPS mutant expressed fimbriae and OMVs and lacked O-polysaccharide chains (Figure 1b). The E69 LPS smooth mutant strain expressed O-polysaccharide chains of LPS, which appeared to bleb off the surface of the bacterium in a different manner to that of OMVs (Figure 1c) and was not observed for the WT strain (Figure 1a). The E69 flagella mutant E69 DfliC did not express any flagella (Figure 1d). In fact, this strain had a strong resemblance to the E69 smooth mutant strain, in that it too had O-polysaccharide chains of LPS, which could be seen to bleb off the bacterial surface. The E69 flagella enhanced strain (fliC+) was found to express lophotrichous flagella and OMVs (Figure 1e). The EAEC WT serotype O42 expressed OMVs and flagella, and formed colonies resembling a stacked brick formation (Figure 2a). The O42 DfliC mutant strain did not express flagella, but did express fimbriae and OMVs, forming colonies in the characteristic stacked brick formation (Figure 2b). The aggregative adherence fimbrial adhesin mutant AafB expressed OMVs only E. Coli-Epithelial Contact Is Key to Host Response (Figure 2c). Another aggregative adherence fimbria mutant AAF/ II expressed a rope like exopolysaccharide and OMVs, but did not express fimbriae (Figure 2d). Collectively, the majority of strains investigated expressed multiple microbial structural components known to interact with host PPRs inducing antimicrobial responses. We therefore went on to investigate the effects of several of the surface structures on the host response. Recognition of EPEC and EAEC flagella contributes to the IEC innate immune response HEp-2 cells respond to apical stimulation with flagellated E69. As E69 flagellin the subunit of flagella has been reported to induce IL-8 secretion by IECs [12] we determined the response to flagella in our model system. Initially, we investigated the effects of apical stimulation with flagellated bacteria, as it is likely that this will be the first interaction between E69 and intact ‘‘healthy’’ mucosa. Viable E69 WT and E69 isogenic flagella enhanced (fliC+) and non flagellated (DfliC) mutants were investigated. At 4 h, it was only E69 that induced any significant secretion of IL-8 (Figure 3a). None of the strains induced any significant CCL20 protein in HEp-2 cells at 4 h (Figure 3b). At 16 h the E69 WT and the two flagella mutants induced a significant increase in IL-8 protein (Figure 3a). Only the WT strain E69 induced significant production of CCL20 protein at this time point (Figure 3b). A statistically significant reduction in IL- 8 expression between the WT strain and the E69 DfliC mutant was observed, indicating that flagella may play a role in apical HEp-2 cell recognition and immune response. There was no significant difference in the induction of CCL20 between the WT strain and the E69 DfliC flagella deficient strain (Figure 3b). The flagella enhanced strain E69 fliC+ showed a more significant reduction in IL-8 and CCL20 in comparison to the WT than the E69 DfliC flagella deficient strain (Figure 3a & b). It has previously been suggested that EPEC has the ability to secrete proteins via its flagella apparatus, which may modulate IEC host responses [15]. HEp-2 cells respond to apical stimulation with flagellated O42. We next investigated the response of HEp-2 cells to apical stimulation with the EAEC WT strain O42 and its isogenic flagella mutant O42 DfliC was investigated. At 16 h the O42 WT and the flagella mutant both induced a significant amount of IL-8 and CCL20 protein (Figure 3c & d). Indeed, stimulation with the O42 WT induced significantly higher levels of the pro-inflammatory cytokines in comparison to stimulation of HEp-2 cells with the E69 WT (Figure 3a & b). The flagella negative mutant showed reduced ability to induce IL-8 and CCL20 protein when compared to the WT strain, implying that flagella are involved in apical HEp-2 cell recognition and response to O42. Caco-2 cells respond to apical stimulation with flagellated E69 and O42. We examined the response of Caco-2 cells to apical stimulation with the EPEC WT strain E69, EAEC WT strain O42 and their respective flagella negative mutant strains E69 DfliC and O42 DfliC. At 16 h the E69 and O42 WTs and the flagella mutants induced a significant increase in IL-8 protein, although the flagella negative mutants showed a reduced ability to induce IL-8 protein when compared to the WT strain (Figure 3e & g). The reduction seen with the flagella mutants suggest that flagella do play a role in the apical Caco-2 cell response to EPEC and EAEC, as has been previously reported [12,16]. Caco-2 cells are un-responsive to basolateral stimulation with flagellated E69. As TLR5, the receptor for flagellin, is reported to be located on the basolateral membrane [17], the response of Caco-2 cells to basolateral stimulation with the EPEC WT strain E69 and the flagella DfliC mutant strain was investigated. Supernatant was collected from the apical and basolateral compartments. Upon basolateral inoculation for 16 h, PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e27030