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.