induced immunity. Variability in O-antigen similarly affects both responses and signalling pathways in macrophages [19]. O-antigen also contributes to localised adherence of EPEC [38], implicating a potential role for attachment in the host response. We found that the IEC immune response was not completely ablated with the rough mutant, suggesting that LPS is not the only factor likely to be involved in apical response to E69. We hypothesised that pro-inflammatory responses may result from the attachment of EPEC and EAEC to host cells. The striking reduction of IL-8 and CCL20 production in response to BFP mutants identifies BFP as a major determinant of the host inflammatory response. The BFP mutant JPN15 was able to form a TTSS and induce intimate adherence, but inhibited NF-kB responses. Thus, without host recognition of BFP the balance is shifted towards an anti-inflammatory response. In support of this, JPN15 can adhere to human intestinal mucosa and produce the AE lesion [32,39], but does not induce neutrophils to cross the epithelium [40]. Bacterial suppression was also seen with the 31-6- 1(1) mutant, but this strain was unable to form intimate adherence. Suppression seen with this strain is potentially TTSS independent, and we found that the EPEC WT E69 down-regulated the host response via secretion of lymphostatin. E69 in the presence of mannose induced the greatest increase in IL-8 and CCL20 protein. Although mannose inhibits adherence via type I fimbriae, E69 can still bind to the epithelia via a variety of additional mechanisms such as BFP, flagella and EspA. The augmentation with mannose suggests that its binding to E69 may act as a MAMP. There have been conflicting reports of the role of the TTSS in host pro- and anti-inflammatory responses [2,4,28]. Our data indicate that both the structural component of the TTSS and effector proteins are involved in the pro-inflammatory responses to E69. The secreted effector protein EspC was involved in host recognition, while EspF did not modulate IL-8 and CCL20 responses. The DlifA strain induced increased IL-8 mRNA expression and CCL20 secretion compared to that of the WT strain. As lymphostatin inhibits peripheral blood and mucosal lymphocyte proliferation and cytokine synthesis [31], it may similarly inhibit epithelial responses. The lifA gene of atypical EPEC is indeed strongly associated with diarrhoea, supporting its role in virulence [41]. We noted a degree of variability in IL-8 mRNA correlation with protein secretion, as has been previously reported and is likely due to post-translational or transcriptional control mechanisms [42]. Despite the variations, similar trends in mRNA and protein suppression via bacterial-epithelial contact (with BFP, flagella or EspA) were found, indicating, by whatever mechanism, the suppression has affected these control mechanisms. IECs thus recognise a combination of EPEC E69 PRR agonists. Host- bacterial contact is significant, as are flagella, LPS, EspA, EspC and the TTSS. In different model systems, EPEC flagella with either EscN [12] or intimin [43] augment host response. EPEC intimate adherence also modulates TLR5 localization and host signalling [43], further supporting the importance of host-bacterial contact. The EAEC WT O42 also induced strong expression of IL-8 and CCL20; indeed greater than the E69 WT, consistent with its greater in-vivo propensity to induce inflammatory diarrhoea [6,7]. Although EPEC can suppress the immune response via the TTSS, EAEC does not do this, but induces an inflammatory response likely to subvert the epithelial barrier [44]. These findings provide insight into the enhanced severity of disease caused by Shiga toxin producing EAEC (O104:H4) during the recent German epidemic [8], in comparison to classic EHEC infection, as systemic E. Coli-Epithelial Contact Is Key to Host Response dissemination of Shiga toxin is likely to be promoted by the increased ability of EAEC to induce an epithelial pro-inflamma- tory and chemokine response. Our data suggest that flagella are involved in apical HEp-2 and Caco-2 cell recognition and response to O42, although the epithelial response to the O42 DfliC mutant was not completely eliminated, and other factors are likely involved. We hypothesised that pro-inflammatory responses may result from attachment of EAEC to host cells in their distinctive aggregative adherence, with a ‘stacked brick-like’ pattern [9], which is key to pathogenesis. Our data indicated that adherence via AAF indeed contributes to IL-8 and CCL20 expression. However, the response is not completely abolished without adherence and recognition of flagella also played a role in host recognition of EAEC O42. This extends previous studies to show that EAEC has a heterogeneous ability to modulate IL-8 [13,45]. Our data provide findings distinct from previous studies using isolated bacterial components such as flagellin monomer. For both EPEC E69 and EAEC O42, isogenic mutants depleted in various structures induced, reduced responses in comparison to the WT. These were however, rarely fully ablated, suggesting a response to more than one component. These data concord with previous findings that factors other than flagellin monomers, including EPEC TTSS and intimate adhesion [12] and EAEC bacterial pAA plasmid [13], impact on the host response. Many non- pathogenic bacteria express flagella and readily release flagellin monomers, and the immune system may thus encounter flagellin more frequently than other bacterial products such as LPS [46]. It would be undesirable for the host to mount a response solely on the presence of flagellin, more likely derived from gut commensals than pathogens. Here, we show that the host response to the whole flagellated bacteria differs to that of the flagellin monomer and the overall innate immune response is due to the recognition of groups of external structures. Hedlund et al. also suggest the host ‘sees’ microbial products not as purified molecules, but as complexes [47]. TLRs do not function in isolation, but form multi-receptor complexes with other PRRs in membrane lipid rafts. Ligand- induced PRR oligomerization modifies the arrangement of TIR domains, altering the binding specificity required for recruitment of appropriate adaptors, allowing combinational diversity in PRR signal transduction pathways [48]. The PRR responsible for detection of type I fimbriae, which bind to mannose, is TLR4 [49]. It would be of interest to determine the PRRs responsible for the recognition of BFP, EscN and the TTSS. The fact that the host responds to combinations of virulence factors has important implications, as failure to develop effective vaccines for complex pathogens may relate to the fact that vaccines to date are mainly to one virulence factor, while simultaneous disruption of multiple virulence factors may be required [50]. Our data support this hypothesis. Adherence was a strong determinant in induction of the IL-8 and CCL20 response to E69 and O42. It may be that such contact dependence is due to the requirement for docking to the cell, allowing lipid rafts to form and recognition of several external structures, thus providing the necessary PRR oligomerization to induce a response. EPEC intimate attachment has indeed been demonstrated through lipid rafts, activating NF-kB and MAPK and production of IL-8 [51]. Co-evolved symbiont bacteria are limited from epithelial contact by IgA coating, antimicrobial peptide secretion and the mucus layer. These bacteria only become a threat following epithelial damage or reduced barrier defence, when they gain access to the epithelium and induce an immune response [42,52]. Whilst our findings are based on study of the prototypical exemplars of EPEC [53] and EAEC [54], PLoS ONE | www.plosone.org 12 October 2011 | Volume 6 | Issue 10 | e27030