Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion
Fig 3
Visualization of T3SS toxin injection in epithelial monolayers.
(A) The bacterial transmigration kinetics is shown as in Fig 1C and compared to cell retraction (Fig 1D), owing to toxin injection, measured on the same dataset (means + SEM, n = 10 fields). Cell retraction was consistently delayed compared to bacterial transmigration in each field. (B) Diagram showing the experimental system used in (C) to monitor T3SS toxin injection: bacteria injecting an ExoS-Bla chimeric toxin were used to infect MDCK monolayers loaded with CCF2, a bifluorescent Bla substrate (see SI Materials and Methods). Once CCF2 is cleaved by ExoS-Bla, fluorescence is shifted from green to blue. Bacterial aggregates at apical surface are represented in yellow, and bacteria in the basal compartment are in red. (C) Top: merged wide-field images at different time points post-CCF2 loading, centered at a transmigration site. Scale bar: 50 μm. Images are selected frames from S5 Movie. Middle: images showing only the blue channel (injected cells). A blue line indicates the external limit of injected cells. Bottom: images showing the bacteria in the basal compartment. The red line represents the outermost bacteria positions and the blue line is as above. (D) Histogram showing the percentage of bacteria within the injected cell surface in 12 fields at 4 h (MOI 30), and within the injected cell surface dilated of 15 μm on each side.
doi: https://doi.org/10.1371/journal.ppat.1005377.g003