Fig 1.
P. aeruginosa aggregates at multicellular junctions.
Cell junction types and P. aeruginosa adhesion sites were analyzed by quantitative confocal microscopy. (A) Representative micrograph showing different cell junctions types: Junction of five cells (full arrowhead), junction of four cells (arrow), junction of three cells (dotted square) and junction of two cells (dotted ellipse). The numbers indicate the cells that come together to form a junction of five. (B) The gray bars (left, y-axis) represent the frequency of junction types, based on the analysis of four randomly imaged regions (~300 cells/region) of a polarized MDCK monolayer. The black circles (right, y-axis) represent the number of P. aeruginosa aggregates formed per junction type normalized to the junction type frequency. Data are presented as mean ± SEM. Pearson correlation coefficient r = 0.95, p<0.05. (C) Projected confocal Z stacks showing examples of P. aeruginosa adhered to multicellular junctions formed by four (left) and five cells (right). PAK-GFP: green and F-actin: red. Scale bars: 10 μm.
Fig 2.
P. aeruginosa adheres to extruded apoptotic cells.
(A) Projected confocal Z stacks and orthogonal sections (lower images) of transwell-grown monolayers infected with PAK. P. aeruginosa adhered to an extruded apoptotic cell (left) and to an apoptotic body (right). PAK-GFP: green, F-actin: red and Annexin V: blue. (B) Glass-grown monolayers infected with PAK. Apoptotic cells to which P. aeruginosa adheres have active Caspase 3. PAK-mCherry: red, Annexin V: green and active Caspase-3: blue. Brightfield depicts the monolayer underneath. (C) Percentage of aggregates formed at Annexin V-positive sites. (D) Percentage of Annexin V-positive spots with at least one adhered bacterium. (E) Percentage of aggregates formed at active Caspase 3-positive sites. Data are presented as mean ± SEM. *p<0.05, Student’s t-test. Scale bars: 10 μm.
Fig 3.
P. aeruginosa adheres to both apoptotic and necrotic cells.
(A) Representative micrographs showing orthogonal sections of apoptotic cell stages and percentage of bacteria adhered to each stage. Data were normalized to the frequency of each apoptotic cell stage. F-actin: red, TO-PRO3 (DNA stain): blue, Annexin V: green. (B) Apoptotic cells extruded from glass-grown MDCK monolayers were stained with Annexin V-Alexa 488 (green). UV-generated apoptotic cells stained with Annexin V-Alexa 647 (blue) were then added to monolayers right before infection with PAK-mCherry (red). (C) H2O2-generated necrotic cells were stained with Annexin V-Alexa 488 (green) and added to glass-grown MDCK monolayers right before PAK-mCherry infection (red). B and C show projected confocal Z stacks. Scale bars: 10 μm.
Fig 4.
P. aeruginosa is internalized through efferocytosis.
Lifeact-GFP MDCK monolayers (green) were stained with Annexin V-Alexa 647 (blue), infected with PAK-mCherry (red) and incubated for 3 h (A and left micrograph in B). Alternatively, UV-irradiated Annexin V-stained wt MDCK cells were added to lifeact-GFP MDCK monolayers followed by PAK-mCherry infection and incubation for 3 h (B, right micrograph). Scale bars: 5 μm. (A) Top view and orthogonal section of a membrane protrusion extending over an apoptotic cell with adhered bacteria. (B) Schemes describing the strategies and corresponding confocal xy planes (upper images) and orthogonal views (lower images) of efferocytic vesicles containing both apoptotic cell debris and bacteria. (C) Transmission electron microscopy of an intracellular membrane vesicle containing several bacteria. Wt MDCK cells monolayers were infected with wt PAK for 3 h. Scale bar: 500 nm.
Fig 5.
Expression of dominant negative Rac1 and preincubation with unlabeled Annexin V inhibit P. aeruginosa internalization.
(A) Proportion of internalized apoptotic material (Intracellular/Total cell-associated apoptotic material). (B) Proportion of internalized P. aeruginosa (Internalized/Total cell-associated bacteria). (A and B) Apoptotic cells generated by UV irradiation were stained with Annexin V-Alexa 647 and added to MDCK-Rac1-N17 filter-grown monolayers and infected with PAK-GFP. The cell-associated apoptotic material and cell-associated bacteria were quantified by image analysis as described in M&M. Dox+ (control) Dox- (dominant negative Rac1). Data were normalized to control. (C) Proportion of internalized apoptotic material after pre-incubating CellTrace-labeled apoptotic cells with unlabeled Annexin V or with binding buffer alone (control) and adding them to transwell-grown lifeact-GFP MDCK monolayers. (D) Proportion of internalized P. aeruginosa after pre-incubating transwell-grown lifeact-GFP MDCK monolayers with unlabeled Annexin V for 15 min in binding buffer. (E) Transwell-grown MDCK monolayers were infected with P. aeruginosa and internalization was measured by standard antibiotic protection assays. When indicated, apoptotic cells from overgrown cultures, pre-incubated with binding buffer alone or with unlabeled-Annexin V, were added to monolayers right before infection. The mean of colony forming units (CFUs) ± SEM was calculated. Data were normalized to control. *p<0.05 vs. control, one-sample t-test.
Fig 6.
P. aeruginosa efferocytic phagosome acquires lysosomal features.
(A) Projected confocal Z stack (top) and orthogonal section (bottom) showing LAMP1-positive vesicles containing bacteria. F-actin: blue, PAK-GFP: green and LAMP1: red. (B) Percentage of intracellular P. aeruginosa found within LAMP1 vesicles over time. (C) Projected confocal Z stack (top) and orthogonal section (bottom) showing acidic vesicles containing bacteria. F-actin: blue, PAK-GFP: green and LysoTracker: red. (D) Percentage of intracellular P. aeruginosa found within acidic vesicles over time. (E) Intracellular P. aeruginosa survival over time. The mean of CFUs ± SEM was calculated. Data were normalized to time 3 h. Scale bars: 5 μm.