E. Coli-Epithelial Contact Is Key to Host Response Figure 4. The O-antigen of LPS contributes to the IEC innate immune responses. HEp-2 cells were either co-cultured with E69, E69 smooth LPS or E69 rough LPS (MOI = 60), or stimulated with IL-1b (10 ng/ml) as a positive control. 4 or 16 h post-infection cell and bacterial co-culture supernatants were harvested and IL-8 (a) and CCL20 (b) protein levels evaluated by ELISA. Data shown is mean (+SD,*/+ P#0.05) induction of three independent experiments. doi:10.1371/journal.pone.0027030.g004 and colonic explants [25]. A fimbriae negative strain (O42 DAAF/ II isogenic mutant) and adhesin negative strain (O42 DAafB isogenic mutant) were utilised. At 4 h there was no statistically significant induction of IL-8 and CCL20 protein expression (Figure 5c & d). However, at 16 h all strains induced a statistically significant secretion of IL-8 and CCL20 (Figure 5c & d); both mutant strains reduced the response in comparison to the WT strain. This suggests that adherence of O42 via AAF and the AAFB adhesin does contribute to IL-8 and CCL20 expression. Recognition of structural and effector components of the EPEC TTSS contributes to the host innate immune response Our data illustrates that the first stage of pathogenesis of EPEC and EAEC, localised adherence to IECs (EPEC via BFP, EAEC via AAF/II), is key to the host pro-inflammatory response. Interest- ingly however, the second stage of EPEC infection is characterised by the TTSS, in a contact dependant manner, injecting effector proteins into IECs, which can result in an anti-inflammatory response [4]. Therefore, it was of interest to investigate the effect of bacterial contact via the TTSS and its effectors on the host pro- inflammatory response in our system. Briefly, in the second stage of EPEC infection EscF binds to EspA, the structural needle protein, and forms a 0.7 mm long hollow extension of the TTSS needle complex. This is made up of polymorphic EspA filaments, through which the EspB and EspD passes before inserting into the host plasma membrane to form a translocation-pore, thus allowing translocation of LEE encoded effector proteins into the host cell cytosol to interfere with signalling processes [26]. EscN a functionally unique ATPase provides an inner-membrane recognition gate for the TTSS chaperon-virulence effector complexes as well as a source of energy for their subsequent secretion [27]. At this stage what remains unclear is the contribution of the secreted effector proteins versus that of the syringe apparatus itself in eliciting an immune response, as there are several contradictory reports in the literature. For example, Sharma et al. indicate that a functional TTSS is required for an anti-inflammatory response [4]. In contrast, other authors have reported that the TTSS is necessary for the activation of MAPK pathways and IL-8 production, while a minimal role for the EPEC TTSS has also been reported [28]. To investigate the potential role of the structural components of the TTSS in IEC immune response(s) to EPEC, the isogenic mutant strains DespA and DescN were used. The DespA strain does not express the needle portion of the TTSS, but does diffusely secrete effector proteins. The DescN strain does not express a TTSS nor does it secrete effector proteins. At 4 h, only E69 induced a modest, but significant expression of IL-8 protein (Figure 6a). A reduction was noted during infection with both TTSS mutant strains, with no strains modulating CCL20 production at this time (Figure 6b). At 16 h all strains induced a significant expression of IL-8 and CCL20 protein (Figure 6a & b). Stimulation with E69 DespA gave a greater increase in CCL20 when compared to the WT albeit not statistically significant. The DescN strain caused significant reduction in IL-8 and CCL20 expression when compared to the WT strain (Figure 6a & b). Thus, in the model system employed here, recognition of structural components of the TTSS and more significantly, the effector proteins appear to be involved in the pro-inflammatory responses to E69. EPEC effector proteins can both contribute to or modulate IEC innate immune responses The contribution of the secreted effector proteins in eliciting an immune response was investigated further using DespC, DespF and DlifA isogenic mutant strains. EspC causes cytotoxic effects including cytoskeletal damage to IECs [29]. EspF functions in immune evasion [30]. The lifA gene encodes for a toxin, lympho- statin, a non-TTSS-secreted protein, which inhibits peripheral blood and also human and murine GI lymphocyte proliferation plus cytokine production [31]. Upon inoculation with the DespC strain for 4 h there was a statistically significant reduction in IL-8 protein in comparison to the WT (Figure 7a). No significant induction of CCL20 was seen at this time. At 16 h there was a statistically significant reduction in both IL-8 and CCL20 protein with the DespC strain in comparison to the WT (Figure 7a & b), suggesting involvement of EspC in host recognition. The lymphostatin negative strain induced significant IL-8 and CCL20 secretion at 4 h (Figure 7a & b). In comparison to the WT, CCL20 secretion was PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e27030