1). Total TLR5 was clearly detected in mock-infected cells (fluorescence intensity value of 169.4 ± 56) with significantly more intensity than in FITC-control cells (4.7 ± 0.3). HB101 interaction did not significantly alter total TLR5 detection (160.0 ± 56.5). Neither E2348/69 nor E22 infection changed TLR5 detection (248.4 ± 92.9 for E2348/69 and 271.1 ± 93.4 for E22) (Fig. 1A). These results confirmed that TLR5 expression is not altered by EPEC infection. However, in non-permeabilized cells (TLR5 on the cell Osimertinib research buy surface), we found a clear difference between infected and non-infected cells (Fig. 1B). In mock-infected cells, surface TLR5 detection was low (average fluorescence value of 22.0 ± 0.4), but still higher than

in the FITC-control cells (5.7 ± 0.2). This result indicates that in non-stimulated cells, most TLR5 is in intracellular compartments and poorly represented on the cell surface.

HB101 interaction did not modify surface TLR5 detection (22.2 ± 0.4). Remarkably, in cells infected with EPEC (either E2348/69 or E22), detection of surface TLR5 was clearly superior to the FITC-control and significantly higher than in mock-infected cells (E2348/69 = 76.0 ± 1.4 and E22 = 54.1 ± 1.0). These increases in surface Small molecule library molecular weight TLR5 detection were the very first evidence indicating that EPEC induces TLR5 re-localization and accumulation on the cell surface of infected cells. To understand the relationship between TLR5 re-localization and EPEC virulence factors, we analysed TLR5 localization in HT-29 epithelial cells infected Clostridium perfringens alpha toxin for 4 h with EPEC E22 Δeae, ΔescN, and ΔfliC mutants by flow cytometry (Fig. 1C, D). Total TLR5 detection was not statistically different in cells infected with E22Δeae (245.4 ± 86.8), E22ΔescN (208.7 ± 52.5) and E22ΔfliC (172.6 ± 43.4) from the value for E22 WT-infected cells (Fig. 1C). Interestingly, in the case of surface TLR5 (Fig. 1D), we found a reduced TLR5 detection on cells infected with E22ΔescN (39.0 ± 0.7) or E22ΔfliC (37.7 ± 0.7) than in E22 WT-infected cells (54.1 ± 1.0). However, in E22Δeae-infected

cells (50.2 ± 2.4), detection of surface TLR5 was almost the same as in E22 WT-infected cells. Even so, infection with any E22 strain (wild-type or its isogenic mutants) induced a slight increase in TLR5 surface expression in comparison with mock-infected cells (22.0 ± 0.4). These data indicate that EPEC T3SS and flagellin participate in TLR5 recruitment towards the cell surface, while the participation of intimin appears to be weak or null. To corroborate EPEC-induced TLR5 surface re-localization, we analysed TLR5 localization in immunofluorescence preparations of non-permeabilized cells, treated with HB101, E2348/69, E22 WT, E22Δeae, E22ΔescN, E22ΔespA or E22ΔfliC. Besides surface TLR5 detection, we used the membrane-permeable reagent TO-PRO-3 to stain DNA as a reference for cell localization. Permeabilized cells were used as a control for total TLR5 detection (data not shown).