Increases in permeability were not the result of epithelial cell death, since cells were still present in monolayers after 16 h of infection (Figure learn more 2B). Figure 2 Epithelial tight junctions are disrupted by AIEC infection. MDCK-I monolayers were grown to confluence on 6.5 mm diameter Transwells and then either left uninfected (sham control; Panel A) or infected with AIEC, strain LF82 (Panel B) at a MOI of 100:1 for 16 h. Monolayers were then

washed with PBS and fixed, blocked and incubated with primary rabbit anti-ZO-1 and the appropriate secondary antibody and DAPI. Panel A: Sham Fer-1 mw control cells showed a normal distribution of ZO-1, outlining the intercellular tight junctions. Panel B: AIEC infection resulted in disruption of ZO-1 localization with large gaps between cells (arrows). Approximate original magnifications: × 630. AIEC infection alters the distribution of ZO-1 Sham control MDCK-I cells (Figure 2A) demonstrated a normal distribution of ZO-1, delineating intact apical cellular junction complexes [27]. Consistent with effects on permeability, Selleck TPCA-1 16 h infection of MDCK-I monolayers with AIEC, strain LF82 (Figure 2B) led to profound disruption of ZO-1 with large gaps between cells with punctate and interrupted distribution of ZO-1, indicating disruption of this integral tight junction

protein [28]. Nevertheless, cells in the monolayer remained viable, as demonstrated by the presence of nuclei and maintenance of normal cells shape and morphology. Disruption of MDCK-I monolayers is accompanied by AIEC invasion and bacterial replication Transmission electron microscopy of infected MDCK-I monolayers was used to define the effect of AIEC infection of polarized monolayers. In contrast to sham control epithelial monolayers, which demonstrated tightly placed cells without expanded intercellular spaces (Figure 3A), AIEC-infected MDCK-I monolayers were disordered after 4 h of incubation, with spaces evident between adjacent cells and disruption of intercellular spaces. Loss of cellular Edoxaban polarity was also observed,

as demonstrated by presence of microvilli on the lateral aspect of infected cells. Furthermore, consistent with previous reports [29], multiple bacteria were seen within cells 4 h after infection with effective replication, indicating that these organisms survive within the cytoplasm of epithelial cells (Figure 3B). Extension of bacterial infection to 48 h resulted in profound disruption of the monolayer, with complete separation between cells and terminal changes in cells, including loss of membrane integrity, chromatin condensation and ballooning of mitochondria (Figure 3C). This effect may be the result of bacterial overgrowth after 48 h of infection. Figure 3 AIEC disrupts MDCK-I monolayers and replicates in the cell cytoplasm.