Characterization of ExoS-Bla and ExoY-Bla reporters.

<p><b>A/</b> Secretion profiles of <i>P. aeruginosa</i> strains carrying the reporter fusion ExoS-Bla. Fifteen µL of culture supernatants of either wild-type CHA strain or mutant CHA strains expressing ExoS-Bla grown under T3SS-inducing conditions were analyzed by immunoblotting with antibodies directed against ExoS, PopB, PopD and PcrV. <b>B/</b> Co-cultures of A549 cells with <i>P. aeruginosa</i> strains for 3 h at MOI 10. Injection of either ExoS-Bla or ExoY-Bla fusions by strains CHA and PAO1ΔSTY was detected after incubating cells with β-lactamase substrate CCF2-AM either by fluorescence microscopy using a 20× objective (upper panel) or by flow cytometry (lower panel). The horizontal bar in flow-cytometry histograms indicates the gating used to determine the percentage of β-lactamase positive cells revealing ExoS-Bla injection. Mean Fluorescence Intensity is indicated in each panel. Scale bar, 100 µm.