Interaction with non-pathogenic E. coli HB101 did not induce localization of TLR5 on the cell surface (Figure S2). These results are consistent with the FACS experiments, where almost all TLR5 was located in intracellular compartments. In contrast, in cells infected with EPEC strains, E2348/69 and E22, TLR5 was clearly detected on the cell
surface (Figure S2). These results confirmed that EPEC infection induces TLR5 re-localization towards the cell surface. Infection with any of the E22 mutant was unable to provoke TLR5 detection on the epithelial cell surface (Figure S2). These results indicate that EPEC T3SS and flagellum participate in the re-localization of TLR5 towards the cellular surface. Notably, in these assays intimin appeared to be necessary KU-57788 for the re-localization of TLR5, a more obvious result than the one obtained with FACS. To know if the localization of another receptor besides TLR5 is altered during EPEC infection, we inquired about TLR4 subcellular
distribution in non-infected cells and in cells infected with E2348/69 during 4 h by examination of immunofluorescent preparations (Figure S3). In mock cells, we found TLR4 equivalent signal intensity and distribution selleck chemicals in permeabilized and in non-permeabilized cells (total and surface TLR4). This indicates that TLR4 is mainly located at the surface of HT-29 cells, which was also true for E2348/69 cells. Therefore, EPEC infection does not affect TLR4 distribution, unlike TLR5 recruitment to the cell surface that was induced by EPEC infection. ERK1/2 signalling pathway (phosphorylation and nuclear translocation) is an important activator of cellular proinflammatory responses. ERK1/2 phosphorylation during EPEC infection (at 2 or 4 h) was detected by WB. Phosphorylated ERK1/2 was not detected in mock-treated cells (normalized band intensity value of 0.026 ± 0.045). HB101 interaction
induced phosphorylation of ERK1/2 (0.673 ± 0.108) but only until 4 h post-interaction. However, in EPEC-infected cells, p-ERK1/2 was clearly detected (Fig. 2A). At 2 h post-infection, both EPEC strains caused equivalent phosphorylation of ERK1/2 (0.737 ± 0.246 for E2348/69 and 0.741 ± 0.064 for E22 infection). However, at 4 h, p-ERK1/2 was stronger during E22 infection (E2348/69: 0.643 ± 0.089 and E22: SDHB 1.01 ± 0.126). Therefore, we confirmed that ERK1/2 phosphorylation in epithelial cells is caused by EPEC E2348/69 infection and found that it was also true for E22. To understand the role of EPEC virulence factors on the phosphorylation of ERK1/2, we performed WB analysis of lysates from cells infected for 4 h with the isogenic EPEC mutants E22 Δeae, ΔescN, ΔespA or ΔfliC (Fig. 2B). Cells infected with T3SS mutants induced ERK1/2 phosphorylation at levels not significantly different than the ones produced by WT infection (1.01 ± 0.126); normalized band intensity values were 1.186 ± 0.207 for E22ΔescN and 1.025 ± 0.209 for E22ΔespA.