Figure 1.
Invasion and Replication of L. monocytogenes at Multicellular Junction Sites
Polarized MDCK monolayers on Transwell filters were infected from the apical (A, C–F) or basal (A and B) sides with L. monocytogenes.
(A) Viable CFUs of intracellular L. monocytogenes were determined after gentamicin treatment. Means and standard deviations from quadruplicate samples are shown. Sample groups are significantly different: unpaired t-test p < 0.0001.
(B–D) Three-dimensional reconstructions of confocal immunofluorescence images of the invasion sites. Upper panels are reconstructions of Z-sections.
(B) At 3 h after basal infection, foci of replication were visualized with antibodies to L. monocytogenes (green) and ZO-1 (red). Arrow indicates a multicellular junction site.
(C) At 1 h after apical infection, a representative site of invasion was visualized with antibodies to ZO-1 (blue). To evaluate intracellular versus extracellular bacteria, we performed an inside/outside staining where extracellular adherent L. monocytogenes were stained before permeabilization in green and both intracellular and extracellular bacteria were stained after permeabilization in red. External L. monocytogenes thus appear as a combination of red/green or yellow.
(D) At 3 h after apical infection, sites of replication were visualized with antibodies to L. monocytogenes (green) and ZO-1 (red), and with phalloidin (blue) to show actin comet-tails in association with the cytoplasmic bacteria, inset.
(E) At 5 h after apical infection, foci of replication and spread were visualized with antibodies to ZO-1 (red) and L. monocytogenes (green).
(F) Using the same methodology as in (E), monolayers were infected with ΔactA L. monocytogenes (green), which are capable of cell invasion and intracellular replication but not cell-to-cell spread.
Scale bars 10 μm.
Figure 2.
Internalin A–Dependent Apical Adhesion and Invasion of Polarized Epithelia
(A and B) Polarized MDCK monolayers were infected from the apical side with Wt, ΔinlA, or ΔinlB L. monocytogenes.
(A) Invasion was determined by quantification of viable CFUs of intracellular bacteria after gentamicin treatment. Means and standard deviations from quadruplicate samples are shown. Sample groups are significantly different: one-way analysis of variance p < 0.0001; Bonferroni t- test p < 0.001 for all pairwise analyses.
(B) Adhesion after 10 min of infection. Means and standard deviations of the number of L. monocytogenes adhered per 1,000 cells from triplicate samples are shown.
(C) Confocal microscopy visualization of Wt L. monocytogenes adhered to a monolayer stained with antibodies to L. monocytogenes (green) and ZO-1 (red). Bacteria are found only at multicellular junctions and are conspicuously absent elsewhere.
(D) A higher magnification area demonstrating concentrated adhesion at a multicellular junction. Scale bars 10 μm.
Figure 3.
Tropism of L. monocytogenes for Multicellular Junctions
Cell junction types and L. monocytogenes adhesion sites were analyzed by quantitative confocal microscopy. The grey bars (left, y-axis) represent the frequency of junction types, defined as the number of cells that meet at a junction, based on analysis of three randomly imaged regions (~500 cells/region) of a polarized MDCK monolayer. The black circles (right, y-axis) represent the number of L. monocytogenes adhered per junction type. Pearson correlation coefficient r = 0.95, p = 0.0001. Bottom: examples of adhesion sites shown by confocal microscopy and visualized with antibodies to L. monocytogenes (green) and ZO-1 (red). L. monocytogenes adhered to multicellular junctions with 3, 6, and 9 cells are shown. Scale bar 10 μm.
Figure 4.
Multicellular Junctions Created by Cell Extrusion
(A) Top panels show reorganization of cells around a site of cell extrusion monitored live by time-lapse differential interference contrast microscopy. The extruding cell is colored in purple, and adjacent cells are outlined in black to show changes in the shape and relative location of the cells over time. The cells surrounding the extruded cell are numbered to illustrate their position at the start and after resolution of the multicellular junction. The bottom strip shows the process of extrusion, beginning at hour 1 of observation, and continuing for 40 min; frames at 10-min intervals.
Early (B) and late stages (C) of cell extrusion from a polarized MDCK epithelium were imaged by confocal microscopy after staining the monolayer for apoptotic nuclei with Sytox green and with antibodies to ZO-1 (red).
(D) Staining of all the cell nuclei in the monolayer with toto-3 (blue) illustrates that a missing nucleus is evident at extrusion sites after cell extrusion is completed. Reconstructed Z-section panels (B–D, top) reveal the rearrangement of junctions that occurs during cell extrusion.
Scale bar 10 μm.
Figure 5.
L. monocytogenes Adhesion to Sites of Cell Extrusion
Polarized MDCK monolayers on Transwell filters were infected from the apical side with Wt L. monocytogenes for 10 min.
(A) Extruding cells were stained with Sytox green prior to fixation. Antibodies to L. monocytogenes (red) were used to visualize adhered bacteria, and anti-ZO-1 (blue) antibodies were used to visualize the cell junctions.
(B) Monolayers were stained with antibodies to L. monocytogenes (green) and ZO-1 (red), and with toto-3 (blue), which illustrates missing nuclei at L. monocytogenes adhesion sites.
Scale bars 10 μm.
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.
Figure 7.
L. monocytogenes Invasion of the Intestinal Epithelium at the Villus-Tip Extrusion Zone
(A) Optical longitudinal section through a villus tip from Wt-GFP L. monocytogenes infected rabbit intestinal tissue stained with phalloidin for F-actin (red) and with toto-3 for nuclei (blue). GFP-expressing bacteria are shown in green.
(B) Three-dimensional reconstruction of villus tips viewed from the lumenal side from tissue stained as in (A).
(C) Optical cross-section through three-dimensional reconstruction of a villus tip revealing intracellular L. monocytogenes (green). F-actin (red) is stained with phalloidin. Right: enlarged view of infected cell revealing actin nucleation on the surface of Wt-GFP L. monocytogenes.
(D) Three-dimensional confocal reconstruction of villus tip from tissue stained with antibodies to ZO-1 (red) and with toto-3 for nuclei (blue). Arrow indicates a cell being extruded.
(E) Three-dimensional reconstruction of infected villus tip stained with antibodies to ZO-1 (red), showing a multicellular junction.
Scale bars 10 μm.