Injection of ExoS-Bla into differentiated HL-60 cells is sensitive to pharmacological agents.

<p><b>A/</b> VD3-differentiated HL-60 cells were exposed to 10 µM of cytochalasin D (CytoD), 10 µM of latrunculin B (LtrB) or 50 nM of Wortmannin for 30 min prior and during infection, or to 2 µM of TAT-C3 toxin, 100 µM of LY-294002, 12 µM of genistein, 10 µM of PP2, and 10 µM of PF-573-228 for 120 min prior and during a 3 h infection period at MOI 10 with the PAO1ΔSTY-SBlaR146A strain. The percentage of injection-positive cells was evaluated by flow cytometry. <b>B/</b> Eukaryotic plasma membranes were purified by fractionation on sucrose gradient, after infection of cells pre-treated with inhibitors as above. Proteins were analysed by immunoblotting using anti-PopB. Ni : non-infected VD3-differentiated HL-60 cells, CTRL: infected VD3-differentiated HL-60 cells without inhibitor. <b>C/</b> The ability of PAO1ΔSTY-SBlaR146A strain to secrete ExoS-Bla <i>in vitro</i> was assayed for each inhibitor modifying injection by immunoblotting of total secreted ExoS-Bla protein using the anti-ß-lactamase antibody. Pa: supernatant of T3SS non-induced <i>P.aeruginosa</i>, IPa: supernatant of T3SS induced <i>P. aeruginosa</i> without inhibitor.</p

Efficiency of ExoS-Bla and ExoY-Bla translocation in different cell lines.

<p><b>A/</b> A549 epithelial cells, BJAB, Jurkat, non-differentiated HL-60 cells (HL-60), or HL-60 differentiated into neutrophils, macrophages and monocytes by DMSO, PMA or Vitamin D3 (VD3), respectively, were infected at MOI of 10, for 3 h, with CHAΔS expressing ExoS-BlaR146A and analyzed by flow cytometry. <b>B/</b> HL-60 and U937 were differentiated in monocytes with VD3 (black bars) or not (white bars) and infected at MOI 10 with PAO1ΔSTY strains expressing either ExoS-BlaR146A or ExoY-Bla as described above. The error bars indicate standard deviation (n = 3). <b>C/</b> Non-differentiated HL-60 (thin lane) or HL-60 differentiated in neutrophils (dot line), monocytes (dash line) or macrophage (thick line) were labelled with a FITC conjugated antibody specific for CD11b and analysed by flow cytometry.</p

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.</p

Switching T3SS resistant HL-60 cells to T3SS permissive cells by serum starvation or panning to anti-CD43.

<p>Non-differentiated HL-60 cells were treated as follows: maintained during 15 min in medium supplemented (+ FCS) or not (− FCS) (a), incubated during 5 min in either naive (− anti CD43) or coated wells with a monoclonal anti CD43 (+ anti CD43), first panned in a anti CD43-coated well during 5 min and then transferred to a naïve well, (transferred) (b) and cells were next incubated at MOI 10 with the CHAΔS-SblaR146A strain and analysed by flow cytometry. Non-differentiated HL-60 were incubated during 5 min in a well coated with anti CD43 in the absence (− CytoD) or the presence (+ CytoD) of 10 µM of cytochalasine D for 30 min prior and during infection at MOI 10 with the CHAΔS-SblaR146A strain (c). The error bars indicate standard deviation (n = 3). Insert: schematic drawing of the experiments presented in the histogram (part <b>b</b>).</p