further study is needed to address any possible inter-strain variation and applicability to all EPEC and EAEC serotypes. Although the importance of epithelial contact has been replicated independently with an alternative EAEC serotype O104:H4 during a recent epidemic [55], implicating a generalised phenomena worth consideration in future vaccine design. As well as host-microbial contact being important, another determinant of the response is time. We consistently found IL-8 to be induced earlier than CCL20, pointing to different signalling events. CCL20 induces recruitment of dendritic cell (DC) precursors [56] which in turn can prime either a tolerogenic or an inflammatory immune response by modulating T cell lineage differentiation [57]. Our data suggest that in early infection IL-8 is induced and initial neutrophil recruitment ensues. With persistent bacterial stimulus, CCL20 is also released to recruit DCs and initiate an adaptive immune response. The only strain to induce significant CCL20 at 4 h was the LifA mutant, identifying lymphostatin as a suppressor of early CCL20 production. In conclusion, there are many layers of complexity to the interaction between bacteria and IECs. The ultimate response represents a balance between host activation and microbial suppression. The host ‘sees’ a bacterium by a combination of PRRs, with host-bacterial contact a key determinant of the IEC response. From the microbial perspective the external structures are essential for colonisation. The ability to make contact with the epithelium is also important, enabling the bacteria to down- regulate host responses, for example via the TTSS. The final outcome depends on bacterial surface structures and soluble secreted factors, PRR compartmentalisation and the location and duration of these interactions. Understanding the complexities of the host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis; potentially swaying the balance towards ‘protective’ immunity. Materials and Methods Bacterial culture and induction of virulence factors All bacterial strains (See Table S1) were first cultured on Brain Heart Infusion (BHI) agar plates at 37uC over 24 h. A single colony from this plate was then cultured overnight in 3 ml of BHI broth at 37uC without shaking. To induce expression of virulence genes, overnight cultures were subsequently diluted 1:30 in DMEM; incubated at 37uC and 100 rpm, until an optical density (OD) at A600 of 0.6 was obtained. Growth in DMEM increases the production of LEE encoded virulence factors [58]. For all strains the corresponding colony forming units (CFU) for an OD of 0.6 was quantified via serial dilutions plated on BHI agar plates. Assessment of bacterial surface structures by negative staining Transmission Electron Microscopy (TEM) Negative staining TEM was used to visualise bacterial surface structures. Formvar/carbon coated copper/rhodium 100 mesh grids were coated with an activated bacterial culture for 1 min. The grid was air dried for 1 min and then coated with 1% aqueous ammonium molybdate (Agar Scientific, Stanstead, UK) for 10 s. Grids were air dried and examined using a Philips Transmission Electron Microscope (Philips CM120, Eindhoven, Netherlands) at an accelerating voltage of 80 kV. Each bacterial culture was examined in duplicate on three separate occasions. The size of the surface structures were estimated by direct measurement from printed micrographs using a measuring magnifier (67) fitted with a 20 mm graticule (Polaron, Watford, Hertfordshire UK). Fimbriae and flagella were assessed visually based on previous morpholog- ical reports. Fimbriae were observed as hollow rod-like structures E. Coli-Epithelial Contact Is Key to Host Response and were distinct from flagella. Type I and II fimbriae are 7–8 nm wide; and type III is 4–5 nm wide. Type I, II and III fimbriae can be approximately 0.5–2 mm long and are peritrichous. Type IV fimbriae are 10–20 mm long and are polar. Long polar fimbriae (LPF) are 2–10 mm long [59]. Flagella are not rigid rod structures and are wider (20 nm) and longer (.20 mm) than fimbriae as well as being monotrichous, lophotrichous or peritrichous [15]. Outer membrane vesicles (OMVs) are spherical vesicles composed of a bi-layer membrane with electron-dense luminal contents [14]. Exopolysaccharide (EPS) is a polymer structure with long polysaccharide chains and additional lipid or peptide groups [60]. Cytokine stimulation Recombinant cytokine interleukin-1b (IL-1b) was reconstituted according to the manufacturer’s instructions (Sigma-Aldrich, Poole, UK). A concentration of 10 ng/ml of IL-1b was routinely used. Mannose inhibition of type 1 pili For adhesion tests, 0.5% D-(+)-Mannose (Sigma-Aldrich, Poole, UK) was added to competitively inhibit any adhesion due to mannose-sensitive type 1 pili [23]. Mammalian Caco-2 and HEp-2 cell culture The Caco-2 cell line (ATCC: HTB-37) is widely used as a model system for the study of enterocytic function and as a model of the intestinal barrier [61]. The HEp-2 cell line (ATCC: CCL- 23) is widely used as a model system to study the adhesion properties of human bacterial enteropathogens [62]. ,16106 cells (Caco-2 or HEp-2) were seeded into a 6-well plate maintained at 37uC in a 5% CO2 in complete culture medium [DMEM supplemented with 10% heat inactivated foetal calf serum (FCS), 2 mM L–glutamine and 1% non-essential amino acids (Sigma, Poole, UK)]. Cells were grown until confluency was attained (Caco-2 for 7 days and HEp-2 cells for 48 h). Mammalian and bacterial co-culture Cells were serum-starved overnight prior to stimulation. Cells were inoculated with bacteria grown to logarithmic phase (MOI <60) for 4 or 16 h at 37uC with 5% CO2. 10 ng/ml of IL-1b served as a positive control. The inoculation was carried out in complete culture medium. To prevent bacterial overgrowth 100 mg/ml gentamicin (Sigma-Aldrich, Poole, UK) was added 3 h post-infection [63]. TranswellH inserts (24 mm diameter, 0.4 mm pore size) were coated overnight with Rat tail collagen 10 mg/cm2 (Sigma- Aldrich, Poole, UK). Caco-2 cells were then seeded at a density of ,16106 onto the inserts. Fresh medium was replenished every 2 days for both chambers. The transepithelial electrical resistance (TEER) was monitored with an epithelial tissue Voltohmeter resistance reader (World Precision Instruments, Stevenage, UK). Cells were allowed to differentiate for 14 days, when TEER values of 500–1000 Vcm2 indicative of intact TJ formation were routinely achieved. The wells were then inoculated either apically or basolateraly. Fluorescent-actin staining (FAS) test The FAS test allows visualisation of actin recruitment that occurs beneath bacterial attachment upon intimate adherence of EPEC [32]. Briefly following co-culture, cell monolayers (grown on13 mm glass cover slips) were washed three times with PBS to remove non-adherent bacteria and were then fixed with 4% formalin/PBS pH 7.4 for 20 min at RTu. The cell monolayers PLoS ONE | www.plosone.org 13 October 2011 | Volume 6 | Issue 10 | e27030