Article Figures & Data
Figures
- 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.
- Fig. 2.
Extracellular protein profiles and Western blot analysis of PA103ΔexoUexoT::Tc expressing various effector proteins in trans and their effects on CHO cells. (A) Coomassie blue-stained polyacrylamide gel (11%) of concentrated culture supernatants from strains grown under inducing conditions (growth in the presence of 10 mM NTA). Supernatants are from strains PA103ΔexoUexoT::Tc pUCP18 (lane 1), PA103ΔexoUexoT::Tc pUCPexoS (lane 2), PA103ΔexoUexoT::Tc pUCPexoSE381A (lane 3), PA103ΔexoUexoT::Tc pUCPexoT (lane 4), PA103ΔexoUexoT::Tc pUCPexoY (lane 5), and PA103ΔexoUexoT::Tc pUCPexoYK81M (lane 6). Molecular mass markers (MWM; in kilodaltons) and the relative mobilities of ExoT (53 kDa), ExoS (49 kDa), and ExoY (42 kDa) are indicated. (B) Western blot of a duplicate gel as shown in panel A. A mixture of specific antisera to ExoS-ExoT and ExoY was used, and the bound antibodies were visualized with a peroxidase-labeled secondary antibody. (C) Cellular morphology of CHO cells infected with PA103ΔexoUexoT::Tc expressing various effector proteins in trans. The name of the expression plasmid in each strain is given above the appropriate picture.
- Fig. 3.
Cell- and supernatant-associated ADP-ribosyltransferase activity of PA103ΔexoUexoT::Tc expressing ExoS, ExoSE381A, or ExoT in trans. ADP-ribosyltransferase activity assays were performed on supernatant- and cell-associated samples collected from uninfected CHO cells or CHO cells infected with PA103ΔexoUexoT::Tc pUCP18, PA103ΔexoUexoT::Tc pUCPexoS, PA103ΔexoUexoT::Tc pUCPexoSE381A, or PA103exoUexoT::Tc pUCPexoT. Activity is normalized to CFU and expressed as 10−4 femtomoles of ADPRT (femtomoles of ADP-ribose transferred to soybean trypsin inhibitor).
Tables
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Known type III effector protein(s) expressed or relevant characteristic(s) Reference(s) or source P. aeruginosa strains PA103 ExoT, ExoU B. H. Iglewski PA103ΔexoU ExoT 4 PA103exoT::Tc ExoU 3 PA103ΔexoUexoT::Tc None This study PA103toxA::Ω ExoT, ExoU B. H. Iglewski PA103exsA::Ω None 7 Plasmids pMOBexoT::Tc Allelic replacement vector encoding tetracycline-interruptedexoT 3, 18 pUCP18 pUC-derived cloning vector able to replicate in P. aeruginosa 17 pUCPexoS Encodes the wild-type ADP-ribosyltransferase ExoS (100% activity) 12 pUCPexoSE381A Encodes the noncatalytic mutant ExoSE381A (0.02% activity) This study and 13 pUCPexoT Encodes the ADP-ribosyltransferase ExoT (0.2% activity) 21 pUCPexoY Encodes the adenylate cyclase ExoY 23 pUCPexoYK81M Encodes the noncatalytic mutant ExoYK81M 23 - Table 2.
Biological effects of the various bacterial strains on CHO cells
Strain Effector(s) % Altered morphologya % Trypan blue staineda Decreased viabilityb None (uninfected) 6 ± 2 0 NAc PA103 ExoT, ExoU 53 ± 2 53 ± 2 + PA103exoT::Tc ExoU 46 ± 8 46 ± 8 + PA103toxA::Ωd ExoT, ExoU NDe ND + PA103ΔexoU ExoT 98 ± 2 0 − PA103ΔexoUexoT::Tc None 6 ± 2 0 − PA103exsA::Ω None NCf 0 − PA103ΔexoUexoT::Tc pUCP None 5 0 − PA103ΔexoUexoT::Tc pUCPexoS ExoS 90 ± 3 0 − PA103ΔexoUexoT::Tc pUCPexoSE381A ExoSE381A 99 ± 1 0 − PA103ΔexoUexoT::Tc pUCPexoT ExoT 99 0 − PA103ΔexoUexoT::Tc pUCPexoY ExoY 95 ± 6 0 − PA103ΔexoUexoT::Tc pUCPexoYK81M ExoYK81M 7 ± 4 0 − ↵a Percentage of CHO cells displaying an altered morphology or trypan blue staining via phase-contrast microscopy; results are averaged from photomicrographs taken of three random fields of infected monolayers.
↵b Presence or absence of a significant reduction in cell viability compared to uninfected control cells by an MTT viability assay.
↵c NA, not applicable; uninfected control cells were used as the basis of comparison for the MTT assay.
↵d Control for the MTT assay; a strain used to show that exotoxin A does not mediate the acute cytotoxic response in this tissue culture model.
↵e ND, not done.
↵f NC, no change in cellular morphology relative to the uninfected control.
- Table 3.
Biological effects of P. aeruginosa type III-secreted proteins on CHO cells
Protein Enzymatic activity Effect on CHO cells ExoS ADP-ribosyltransferase Morphological alterations ExoT ADP-ribosyltransferase Morphological alterations ExoU Unknown Acute cytotoxicity: membrane permeability, loss of viability Morphological alterations ExoY Adenylate cyclase Morphological alterations
