Journal: PLoS ONE

Article Title: Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

doi: 10.1371/journal.pone.0030488

Figure Lengend Snippet: A/ 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/ 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. C/ The ability of PAO1ΔSTY-SBlaR146A strain to secrete ExoS-Bla in vitro 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 P.aeruginosa , IPa: supernatant of T3SS induced P. aeruginosa without inhibitor.

Article Snippet: Cells were also exposed to either 100 µM of ROCK inhibitor Y-27632, 2.5 nM of Toxin B from Clostridium difficile (Sigma Chemical Co), 2 µM of TAT-C3 toxin from Clostridium botulinum (Cytoskeleton), 10 µM of PP2, 10 µM of PF-573-228 (Calbiochem), 100 µM of LY-294002 (Sigma Chemical Co) or 12 µM of Genistein (Sigma Chemical Co) for 120 min prior to and during infection in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Infection, Injection, Flow Cytometry, Purification, Fractionation, Western Blot, In Vitro