FIG. 1.

Extracellular protein profiles, Western blot analysis, and infection of CHO cells with parental (PA103) and mutant (PA103ΔexoU, PA103exoT::Tc, and PA103ΔexoUexoT::Tc) strains of P. aeruginosa. (A) Coomassie blue-stained polyacrylamide gel (10%) of concentrated culture supernatants from strains grown in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of 10 mM NTA, a chelator that induces the expression of the exoenzyme S regulon. Supernatant fractions were collected and concentrated 20-fold by the addition of a saturated ammonium sulfate solution to 55% from strains PA103 (lanes 1 and 2), PA103ΔexoU (lanes 3 and 4), PA103exoT::Tc (lanes 5 and 6), and PA103ΔexoUexoT::Tc (lanes 7 and 8). Molecular mass markers (MWM; in kilodaltons) and the relative mobilities of ExoU (72 kDa), ExoT (53 kDa), PcrV (32.2 kDa), and PopD (31 kDa) are indicated. (B) Western blot of a duplicate gel as shown in panel A. A mixture of specific antisera reactive to ExoU, ExoS-ExoT, PcrV, and PopD was used as the primary antibody. Bound antibodies were visualized with a peroxidase-labeled secondary antibody and 4-chloro-1-naphthol and peroxide as substrate. (C) Phase-contrast microscopy (40× objective) of CHO cell morphology following infection with parental or mutant strains of P. aeruginosa. The results of the trypan blue staining for uninfected cells, PA103, and PA103exoT::Tc (10× objective) are shown in the insets. Only infections with bacterial strains expressing ExoU resulted in trypan blue staining 3 to 4 h after bacterial infection.