were permeabilised with 0.1% Triton-X 100/PBS for 30 min. The cells were incubated with 5 mg/ml FITC-conjugated phalloidin (Sigma-Aldridge, Poole, Dorset, UK) for 60 min at RTu followed by washing in PBS three times for 1 min and mounted onto glass slides using citifluor (Agar Scientific, Stanstead, UK). The cells were examined using a Zeiss UV microscope with 406 lens (numerical aperture 1.5) and images were acquired with a Zeiss Axiocam digital camera system (8-bit, 130061300 pixel-standard resolution) (Software-Image Associates, UK). The number of localised or intimate adherent bacteria was quantified. Using inverse thresholding, grayscale images of both phase contrast and fluorescent micrographs were separately binarized. The adherent and intimate adherent bacteria appeared as dots that were quantified using the ‘‘analyze particle’’ tool of ImageJ (National Institutes of Health [NIH], Bethesda, USA). A ratio of adherent bacteria per cell was calculated. The total number of localised/intimate adherent bacteria per micrograph was divided by the total number of HEp-2 cells per micrograph. A total of three fields of view were selected at random for each condition per experiment and three independent experiments were conducted. The micrographs were thresholded under the same conditions. Cytokine specific gene expression Following infection, cell monolayers were subjected to RNA extraction utilising TRIZOL (Invitogen, Paisley, UK) according to manufacturer’s instructions. 5 mg of total RNA was reversed transcribed using the BioScript cDNA synthesis kit (Bioline, London, UK) according to the manufacturer’s instructions. This was followed by RT-PCR, using previously characterised primers for human IL-8 and GAPDH (see Table S2) [64,65]. Bands were visualized by ethidium bromide staining (Amresco, NBS Biolog- icals, Cambs, UK). Densitometric analysis of bands was conducted using the ‘‘gel analyzer’’ tool of ImageJ (NIH) software. Variations in mRNA levels were normalized to GAPDH expression and are expressed as fold induction compared to the uninfected control cells. Cytokine specific protein secretion For the detection of IL-8 or CCL20 secretion into culture supernatants, IL-8 or CCL20 sandwich ELISA kits were used following the manufacturer’s instructions (Quantikine, R&D, Abingdon, UK). Statistics Statistical analyses were performed using SPSS 14 for Windows. Differences in gene or protein expression between control cells and References 1. UNICEF/WHO (2009) Diarrhoea: why children are still dying and what can be done. 2. Ruchaud-Sparagano MH, Maresca M, Kenny B (2007) Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function. Cell Microbiol. 3. Scaletsky IC, Silva ML, Trabulsi LR (1984) Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun 45: 534–536. 4. Sharma R, Tesfay S, Tomson FL, Kanteti RP, Viswanathan VK, et al. (2006) Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 290: G685–G694. 5. Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA (1983) Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 41: 1340–1351. 6. Schuller S, Lucas M, Kaper JB, Giron JA, Phillips AD (2009) The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection. Cell Microbiol 11: 521–530. E. Coli-Epithelial Contact Is Key to Host Response stimulus (denoted as * on reaching statistical significance) and between wild type (WT) strains and their respective mutant strains (denoted as + on reaching statistical significance) were evaluated using a two-tailed Mann-Whitney U-test. Supporting Information Figure S1 EPEC BFP, TTSS and lymphostatin implicat- ed in the balance between host induction and EPEC inhibition of IL-8 mRNA. IL-8 and GAPDH gene expression of HEp-2 cells 4 h post-infection was determined by RT-PCR. Shown is a representative gel. Lane numbers: 1-unstimulated control, 2-IL-1b, 3-E69, 4-E69 + mannose, 5-E69 Smooth LPS, 6-E69 Rough LPS, 7-E69 DfliC, 8-E69 fliC+, 9-E69 31-6-1(1), 10-E69 JPN15, 11-E69 DecsN, 12-E69 DespA, 13-E69 DespF, 14- E69 DespC, 15-E69 DlifA, 16-mannose (17-reverse-transcriptase omitted). (TIF) Figure S2 Phase-contrast and fluorescence micrographs showing control HEp-2 cells (a) and HEp-2 cells co- cultured with E69 WT (b) at 16 h. Utilizing the fluorescent- actin staining test, each co-culture was examined in triplicate, shown is a representative of those seen. Complementarities of bacterial location [white arrow] and actin fluorescence [yellow arrow] was confirmed by simultaneously recording phase-contrast and fluorescence images. The images were analysed and the number of adherent bacteria quantified (see Figure 9). (TIF) Enteropathogenic E. coli [EPEC] species. Primers utilized in this study. Table S1 (DOC) Table S2 (DOC) Acknowledgments We would like to thank Professor Brett B Finlay, Professor James B Kaper, Professor James P Nataro, Professor Michael S Donnenberg and Professor Ilam Rosenshine for the provision of the bacterial species. Many thanks also to Dr. Kristen Knack and Dr. Stephanie Schu¨ ller for their technical guidance. Author Contributions Conceived and designed the experiments: ADP SHM LAE. Performed the experiments: LAE. Analyzed the data: LAE. Contributed reagents/ materials/analysis tools: ADP SHM. Wrote the paper: LAE. Intellectual content: NJK MBE. 7. 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(2008) Flagellin-dependent and -independent inflammatory responses following infec- PLoS ONE | www.plosone.org 14 October 2011 | Volume 6 | Issue 10 | e27030