E. Coli-Epithelial Contact Is Key to Host Response Figure 8. EPEC BFP, TTSS and lymphostatin implicated in the balance between host induction and EPEC inhibition of IL-8 mRNA. IL- 8 gene expression of HEp-2 cells 4 h post-infection was determined and normalised to GAPDH. Variations in mRNA levels are expressed as fold induction compared to the uninfected control cells. Data shown is mean (+SD,*/+ P#0.05) induction of three independent experiments. A representative gel is included (see Figure S1). doi:10.1371/journal.pone.0027030.g008 significant reduction in expression compared to the WT and suggesting that recognition of EspA contributes to the host pro- inflammatory response. In addition, flagella may be involved in suppression of the host response. E69 WT incubated in the presence of mannose induced a significant increase in IL-8 mRNA, not seen with stimulation of mannose alone. Upon infection with the BFP mutant strains JPN15 and 31-6-1(1), levels of IL-8 mRNA were found to be even lower than the ‘constitutive’ expression noted in uninfected control cells and were significantly reduced in comparison to the WT strain. Therefore, on removal of the external bacterial structure BFP the host inflammatory response is not stimulated and a bacterial mediated anti- inflammatory response then outweighs the host pro-inflammatory response to EPEC. There was significant induction of IL-8 mRNA expression with the DescN, DespC and DespF strains, but no significant difference in expression compared to the WT was observed. The lymphostatin negative strain induced statistically significant expression of IL-8 mRNA compared to the WT; suggesting that lymphostatin inhibits IL-8 mRNA expression. Overall our data suggest that there is a delicate balance between the host pro-inflammatory response and EPEC suppression of this response. Contribution of the EPEC intimate adherence to IEC immune response As our data demonstrates that localised adherence to IECs is key determinant of the host pro-inflammatory response to EPEC it was important to investigate the third stage of EPEC infection characterised by intimate adherence to the mucosal membrane forming an attaching and effacing lesion. In this series of experiments the intimate adherence status of the EPEC strains were investigated using the FAS test. Ultra structural studies show the accumulation of cytoskeletal actin, beneath intimately attached bacteria. Knutton et al. developed a specific fluorescent-actin staining (FAS) test, which is diagnostic for the AE adherence property [32]. The nucleation of actin by bacteria to form filamentous or F-actin, which is stained by FITC conjugated phalloidin, allows the bacteria to be divided into fluorescent actin staining (FAS) positive (intimately-adherent) or FAS negative bacteria (non-adherent). The control, uninfected HEp-2 cells at 16 h showed a typical negative FAS test, with actin fluorescence localised at the cell periphery (Figure S2). Upon infection with E69 WT the FAS test was positive, with fluorescence localizing at the cell periphery in addition to intense spots of actin fluorescence, which in PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e27030