Invasion and Replication of <i>L. monocytogenes</i> at Multicellular Junction Sites

<div><p>Polarized MDCK monolayers on Transwell filters were infected from the apical (A, C–F) or basal (A and B) sides with <i>L. monocytogenes</i>.</p><p>(A) Viable CFUs of intracellular <i>L. monocytogenes</i> were determined after gentamicin treatment. Means and standard deviations from quadruplicate samples are shown. Sample groups are significantly different: unpaired <i>t-</i>test <i>p</i> < 0.0001.</p><p>(B–D) Three-dimensional reconstructions of confocal immunofluorescence images of the invasion sites. Upper panels are reconstructions of Z-sections.</p><p>(B) At 3 h after basal infection, foci of replication were visualized with antibodies to <i>L. monocytogenes</i> (green) and ZO-1 (red). Arrow indicates a multicellular junction site.</p><p>(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 <i>L. monocytogenes</i> were stained before permeabilization in green and both intracellular and extracellular bacteria were stained after permeabilization in red. External <i>L. monocytogenes</i> thus appear as a combination of red/green or yellow.</p><p>(D) At 3 h after apical infection, sites of replication were visualized with antibodies to <i>L. monocytogenes</i> (green) and ZO-1 (red), and with phalloidin (blue) to show actin comet-tails in association with the cytoplasmic bacteria, inset.</p><p>(E) At 5 h after apical infection, foci of replication and spread were visualized with antibodies to ZO-1 (red) and <i>L. monocytogenes</i> (green).</p><p>(F) Using the same methodology as in (E), monolayers were infected with Δ<i>actA L. monocytogenes</i> (green), which are capable of cell invasion and intracellular replication but not cell-to-cell spread.</p><p>Scale bars 10 μm.</p>