The new eae sequences of strains analyzed were deposited in the European Bioinformatics Institute (EMBL Nucleotide Sequence Database). Quantitative invasion assay Quantitative assessment of bacterial invasion was performed as described previously [53] with modifications. Briefly, washed HeLa and polarized and differentiated T84 cells were infected with 107 colony-forming click here units (c.f.u.) of each aEPEC strain for 6 h or 3 h for tEPEC E2348/69. The different incubation-periods used were due to the more

efficient colonization of tEPEC in comparison with the aEPEC strains; moreover, tEPEC E2348/69 induced cell-detachment in 6 h. Thereafter, cell monolayers were washed five times with PBS, and lysed in 1% Triton X-100 for 30 min at 37°C. Following cell lysis, bacteria were re-suspended in PBS and quantified by plating serial dilutions onto MacConkey agar plates to obtain the total number of cell-associated bacteria (TB). To obtain the number of intracellular bacteria (IB), a Selleckchem ACP-196 second set of infected wells was washed five times and 4SC-202 further incubated in fresh media with 100 μg/mL of gentamicin for one hour. Following this incubation period, cells were washed five times, lysed with 1% Triton X-100 and re-suspended in PBS for quantification by plating serial dilutions. The invasion indexes were calculated as the percentage of the total number of cell-associated bacteria (TB) that

was located in the intracellular compartment (IB) after 6 h (or 3 h for tEPEC E2348/69) (IBx100/TB) of infection. Assays were carried out in duplicate, and the results from at least three independent experiments were expressed as the percentage of invasion Cyclic nucleotide phosphodiesterase (mean ± standard error). Cytoskeleton polymerization inhibitor In order

to evaluate the participation of cytoskeleton components in the invasion of aEPEC 1551-2, HeLa cell monolayers were incubated with 1 and 5 μg/mL of Cytochalasin-D or Colchicine (Sigma-Aldrich, St. Louis, MO) 60 min prior to bacterial inoculation [33]. After that, cells were washed three times with PBS and the invasion assay was performed as described above. S. enterica sv Typhimurium and S. flexneri were used as controls. EGTA treatment for tight junction disruption In order to evaluate the interaction of aEPEC 1551-2 with the basolateral surfaces of T84 cells, differentiated cell monolayers (14 days) were incubated with 1 or 5 mM of EGTA (Sigma-Aldrich, St. Louis, MO) 60 min prior to bacterial inoculation [35]. After that, cells were washed three times with PBS and the invasion assay was performed as describe above. S. enterica sv Typhimurium and S. flexneri were used as controls. Detection of actin aggregation To detect actin aggregation the Fluorescence Actin Staining (FAS) assay was performed as described previously [12]. Briefly, cell monolayers were infected for 3 h, washed three times with PBS and incubated for further 3 h with fresh medium.