<p>(A) The <i>P. aeruginosa</i> wild type strain or the one transformed with empty plasmid pUCP20 or pUCP20 containing cDNA encoding for rExoS129-S100A8 or rExoS129-S100A9 were induced <i>in vitro</i> upon calcium depletion by 5 mM EGTA as described in Materials and Methods. Recombinant ExoS129-S100A8 and rExoS129-S100A9 secreted by TTSS in the culture medium, were detected by Western blot with rabbit polyclonal antibodies raised against purified S100 proteins of neutrophils cytosol as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040277#pone.0040277-Paclet1" target="_blank"></a> and in Materials and Methods. Immune complexes were stained by ECL. (B) TTSS properties of <i>P. aeruginosa</i> were induced <i>ex vivo</i> by the contact between CHA strains and EBV-B lymphocytes (MOI 10) in the presence of 10% (v/v) human AB serum (RPMI 1640 medium,) in order to deliver recombinant ExoS-S100 fusion proteins (rExoS129-S100A8, rExoS129-S100A9, rExoS54-S100A8, rExoS54-S100A9, ExoS129-S100A9-A8, ExoS54-S100A9-A8, or ExoS54-S100A8-A9 chimeras). In some experiments, bacteria CHA S100A9 and CHA S100A8 were concomitantly induced by contact for 90 min at 37°C with EBV-B lymphocytes (MOI 5 each) in RPMI 1640 medium. NADPH oxidase activity of 2×10<sup>6</sup> EBV-B lymphocytes, collected after injection of fusion proteins by TTSS, was measured by chemiluminescence, upon stimulation by 130 nM PMA. Activity was expressed as RLU. Number of experiments n = 3 to 5. Data represent means±SD. *p<0.05, significant difference versus control (NADPH oxidase activity of EBV-B lymphocytes after contact with pUCP20 transfected <i>P. aeruginosa</i>). (C) EBV-B lymphocytes, either control cells or cells after rExoS 54-S100A9-A8 chimera injected by <i>P. aeruginosa,</i> were fixed, permeabilized, and labeled with goat polyclonal antibody anti human S100A8, C19 (dilution 1∶1,000) for confocal microscopy analysis, as described in Materials and Methods. An Alexa Fluor 488 anti-goat antibody was used to detect the fusion proteins, stained in green, in cells when rExoS54-S100A9-A8 proteins were present. EBV-B lymphocyte nuclei were stained in blue with Hoechst 33258.</p
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