E. Coli-Epithelial Contact Is Key to Host Response Figure 3. HEp-2 and Caco-2 cells respond to apical stimulation with flagellated EPEC and EAEC; however, Caco-2 cells are un- responsive to basolateral stimulation with flagellated EPEC. Cells were either co-cultured with E69, the E69 flagella mutant DfliC strain, the E69 flagella enhanced strain fliC+, O42 and O42 DfliC (MOI = 60), or stimulated with IL-1b (10 ng/ml) as a positive control. At 4 or 16 h post-infection with E69 or O42, HEp-2 cell and bacterial co-culture supernatants were harvested and IL-8 (a & c) or CCL20 (b & d) evaluated by ELISA. Caco-2 cells were stimulated apically for 16 h and IL-8 protein evaluated (e & g). Caco-2 cells were also stimulated basolaterally for 16 h, apical and basolateral IL-8 secretion was evaluated (f & h). Data shown is mean (+SD,*/+ P#0.05) induction of three independent experiments. doi:10.1371/journal.pone.0027030.g003 measured in uninfected control cells. In comparison, a marked increase in IL-8 protein was observed on stimulation with the flagella mutant. Thus, the aflagellated strain induced a statistically significant increase in IL-8 secretion in comparison to the WT strain both apically and basolaterally (Figure 3f). These data suggest a role of flagella in the inhibition of the host response to basolateral inoculation with EPEC WT E69. Caco-2 cells are responsive to basolateral stimulation with flagellated O42. The response of Caco-2 cells to basolateral stimulation with the EAEC WT strain O42 and the flagella DfliC mutant strain was investigated. Supernatant was collected from the apical and basolateral compartments. Upon basolateral inoculation for 16 h, a modest amount of apical IL-8 secretion was seen with the O42 WT and the flagella negative mutant (Figure 3 h). Minimal IL-8 was detected in the basolateral compartment of control cells with an increase in IL-8 secretion into the basolateral chamber during infection, with the WT strain and the flagella mutant, showing no significant difference (Figure 3 h). These data suggest that flagella are not involved in the host response to basolateral inoculation with EAEC WT O42. Collectively, these data do indicate a role for flagella in apically induced immunity. However, as the IEC responses were not completely ablated on apical stimulation with the flagella mutants in comparison to the WT, we hypothesised that in addition to flagella other bacterial factors may be involved in IEC recognition. Recognition of the O-polysaccharide chains of EPEC lipopolysaccharide (LPS) contributes to the IEC innate immune response LPS is the dominant component of the outer membrane of gram- negative bacteria and is released when bacteria multiply and die [18]. Therefore, the involvement of LPS to host recognition was investigated. LPS consists of a lipid A moiety, a core polysaccharide and O-polysaccharide chains of variable lengths. Colony morphol- ogy is indicative of O-glycosylation status. Smooth colony forming strains express the complete core with varying chain lengths, whereas rough colony forming mutants lack the O-polysaccharide chains [19]. Despite extensive studies the LPS structures (i.e. core or polysaccharide chains) that induce IEC inflammatory responses have not been unequivocally identified. In this series of experiments, the involvement of EPEC WT E69 LPS was investigated with the use of isogenic rough and smooth LPS mutant strains. The TLR4/ MD-2/CD14 receptor complex recognises LPS. Due to the conflicting reports as to whether Caco-2 cells express biologically active TLR4, MD-2 or CD14 [20,21] the host innate immune response to EPEC LPS in HEp-2 cells was established. On exposure to WT E69, but not rough/smooth LPS mutants, a minimal increase in IL-8 protein secretion 4 h post-infection was noted (Figure 4a). However, induction in IL-8 and CCL20 protein in response to all strains was observed 16 h post-bacterial exposure (Figure 4a & b). There was a modest, but not significant increase in CCL20 expression between the WT and smooth LPS isogenic mutant strains (Figure 4b). The rough LPS strain exhibited reduction in its ability to induce both IL-8 and CCL20 protein expression in comparison to the WT (Figure 4a & b). These data are indicative of a role for O-polysaccharide chains in LPS–induced immunity. Significant contribution of adherence to IEC innate immune response Once again, the inflammatory response was not completely ablated upon infection with the LPS mutants, in comparison to the WT bacteria; thus in addition to flagella and LPS other bacterial factors are involved in IEC responses. We hypothesised that pro- inflammatory responses may result from the attachment of EPEC and EAEC to host cells. This is particularly relevant, given that aggregative adherence and intimate attachment to IECs are hallmarks of EAEC and EPEC infection respectively. In the following series of experiments the contribution of adherence to HEp-2 cell responses of EPEC WT strain E69 was investigated. Localised adherence of EPEC. The first stage of EPEC colonisation is a characteristic pattern of adherence called ‘‘localised adherence’’. Several bacterial surface-organelles and secreted products have been implicated in this adherence; such as the bundle forming pilus (BFP), type I fimbriae and EPEC secreted protein A (EspA) [22]. BFP is a type IV fimbriae encoded on a large plasmid called the EPEC adherence factor plasmid (EAF) [9]. The role of BFP was studied with isogenic mutant E. coli strains JPN15 (lacks the EAF plasmid) and 31-6-1(1) (with a TnphoA insertion - an inactivation mutation in the virulence plasmid-encoded bfpA gene). Type I fimbriae adherence was inhibited by the addition of mannose to infections of WT E69 [23]. EspA filaments of the TTSS needle complex adhere EPEC to the host cell [24]. The role of EspA was investigated by using a DespA isogenic mutant. At 4 h the E69 WT and the WT plus mannose caused a modest induction in IL-8 protein (Figure 5a). Strains JPN15 and 31-6-1(1) showed no expression of IL-8 protein. None of the strains investigated induced any significant change in expression of CCL20 protein at 4 h (Figure 5b). At 16 h the E69 WT, the WT plus mannose and the EspA mutant induced a marked increase in IL-8 and CCL20 protein (Figure 5a & b). At 16 h in comparison to the WT, JPN15 and 31-6-1(1) showed a statistically significant reduction in expression of IL-8 and CCL20 protein (Figure 5a & b). The levels of CCL20 protein were found to be even lower than the ‘constitutive’ expression noted in uninfected control cells. Suggesting that recognition of BFP contributes to the host pro- inflammatory response and the inflammatory response is not stimulated in the absence of BFP. Furthermore, as the levels are below the constitutive expression the balance between host and microbe has been tipped in favour of bacterial suppression. E69 plus mannose induced the most statistically significant increase in IL-8 and CCL20 protein amongst the strains tested (Figure 5a & b). There was no significant difference in IL-8 and CCL20 protein expression between the EspA mutant and E69 WT (Figure 5a & b). Collectively these data implicate adherence of E69 via BFP as a strong determinant of IL-8 and CCL20 expression. Aggregative adherence of EAEC. EAEC has a distinctive aggregative adherence, with a ‘stacked brick-like’ pattern dis- tinguishable from that manifested by EPEC [9] as seen in (Figure 2b). EAEC adherence requires expression of aggregative adherence fimbriae (AAFs). EAEC prototype strain O42 expresses the AAF/II allele, encoded on plasmid pAA2. The AAF/II fimbriae is 5 nm in diameter and mediates adhesion to HEp-2 cells PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e27030