Listeria monocytogenes Invades the Epithelial Junctions at Sites of Cell Extrusion
Figure 6
L. monocytogenes Attachment to Accessible E-cadherin at Multicellular Junctions of Cell Extrusion Sites
(A and B) Polarized MDCK monolayers on Transwell filters were incubated with Sytox green prior to fixation. Monolayers were left unpermeabilized and stained from the apical side with an antibody to the extracellular domain of E-cadherin (red) and with phalloidin to visualize the F-actin cytoskeleton (blue).
(A) A site with a cell in the process of extrusion.
(B) A site where extrusion has been completed.
(C) Polarized MDCK monolayers on Transwell filters were infected from the apical side with L. monocytogenes for 10 min, then stained from the apical side with antibodies to L. monocytogenes (green) and E-cadherin (red) without permeabilizing the sample.
(D) Polarized MDCK monolayers on Transwell filters were pretreated with anti-gp135 or anti–E-cadherin antibodies, then infected with L. monocytogenes for 5 min. Means and standard deviations of the number of L. monocytogenes adhered per 1,000 cells from triplicate samples are shown. The E-cadherin antibody–treated sample group is significantly different: one-way analysis of variance p = 0.0004. Bonferroni t-test Mock versus gp135 p > 0.05; Mock or gp135 versus E-cadherin p < 0.001. Shown below the bar graph, blocking antibody concentrations were normalized using a fluorescence-based dot-blot analysis ([Ab] Dot-blot).
(E) Confocal immunofluorescence images show the localization of the blocking antibodies (red), adhered L. monocytogenes (green) and the F-actin cytoskeleton (blue).
Scale bars 10 μm.