comparison with the complementary phase-contrast image, correspond in size and position with adherent bacteria (Figure S2). Thus, the WT strain behaved as previously described [32]. The EPEC E69 flagella negative mutant (DfliC) and flagella enhanced strain (fliC+) were both FAS test positive, as were the EPEC E69 LPS smooth and rough mutant strains (data not shown). This demonstrates that the mutant strains are able to form AE lesions in a manner similar to the WT strain, indicating that these mutations do not alter the strain phenotype or their adhesion status and therefore, any reduction seen with the mutant strains is not due to alteration in intimate adhesion status. The EPEC E69 bundle forming pilus mutant 31-6-1(1), which has a TnphoA in bfpA no longer conferring localised adherence, was FAS test negative, with actin fluorescence localised at the cell periphery (data not shown). These data would suggest that initial localised adherence may influence intimate adherence and the formation of AE lesions. However, the EPEC E69 mutant JPN15 that lacks the EAF plasmid on which BFP is encoded [33], preventing localised adherence via BFP, was weakly FAS test positive, showing characteristic spots of actin fluorescence corresponding in size and position with adherent bacteria (data not shown). This suggests that the JPN15 strain utilises an alternative to BFP for initial localised adherence prior to forming an AE lesions, such as type I fimbriae, EspA or flagella [22]. The TTSS mutant strains DespA and DescN were confirmed as FAS test negative (data not shown). Upon quantification of the number of localised or intimate adherent bacteria using ImageJ software, we found similar levels of adherent bacteria for all strains, with an average of twenty adherent bacteria per cell from an initial inoculate of sixty per cell (Figure 9a). This shows that any reduction in the inflammatory response observed with the mutant strains in comparison to the WT was not due to alterations in the levels of infectivity for the strains, but due to removal of the bacterial surface structures themselves. For the majority of strains an average of ten intimately adherent bacteria per cell was observed. Thus, approximately fifty percent of the adherent bacteria were intimately adhered (Figure 9b), which may explain the overall balance to a pro- inflammatory response; as only half of the adherent bacteria would have the ability to suppress the host response. No intimate adherence was seen with the BFP mutant strain 31-6-1(1), or the E69 DespA and DescN TTSS mutants (Figure 9b). Interestingly, an increased percentage of intimately adhered bacteria were observed with the smooth LPS strain (from 50 to 75%), which may account for the differences observed in IL-8 and CCL20 expression between the WT and smooth LPS isogenic mutant strain. Also, while not statistically different from the WT, a 25% difference in expression of IL-8 and CCL20 is observed with the smooth LPS strain (Figure 4). Thus we found a correlation between the number of intimately adhered bacteria and the level of the host response observed. Discussion This study aimed to characterise the bacterial motifs on EPEC and EAEC that induce epithelial IL-8 and CCL20 responses [34,35]. We investigated the contribution of ‘‘native’’ flagella rather than using flagellin monomers, finding a role in apical HEp- 2 and Caco-2 cell recognition and immune response. This contrasts with reports of basolateral responses to the flagellin subunit [17]. Others have noted differential host responses to flagellated versus aflagellated bacteria and intact flagella versus ‘‘monomeric’’ flagellin protein [36,37]. The E69 DfliC mutant induced greater IL-8 secretion than wild type E69 basolaterally, E. Coli-Epithelial Contact Is Key to Host Response Figure 9. Quantification of localised (a) and intimate (b) adherent E69 at 16 h. Following the fluorescent-actin staining test, complementarity of bacterial location and actin fluorescence was confirmed by simultaneously recording phase-contrast and fluores- cence images. The micrographs were analysed with ImageJ software (NIH) to quantify the number of adherent bacteria per cell. Data shown as mean (+SD) of three independent experiments. doi:10.1371/journal.pone.0027030.g009 suggesting that flagella inhibit host responses at this site, possibly by their secretion of inhibitory effector proteins at the basolateral surface [15]. This is an active process requiring the presence of viable bacteria, which may suppress host responses after epithelial barrier disruption. In support of this, the flagella enhanced strain E69 fliC+ induced greater reduction in IL-8 and CCL20 than the E69 DfliC flagella deficient strain. There is currently no flagella enhanced EAEC O42 strain to determine whether this is a generalised phenomenon. We investigated the role of LPS in apical response, which was maintained in flagella mutants. Our data demonstrated a role for O-polysaccharide chains in LPS– PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e27030