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MOLECULAR AND CELLULAR PATHOGENESIS

Biological Effects of Pseudomonas aeruginosa Type III-Secreted Proteins on CHO Cells

Amy J. Vallis, Viviane Finck-Barbançon, Timothy L. Yahr, Dara W. Frank
Amy J. Vallis
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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Viviane Finck-Barbançon
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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Timothy L. Yahr
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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Dara W. Frank
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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DOI: 10.1128/IAI.67.4.2040-2044.1999
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  • Fig. 1.
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    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.
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    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.
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    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

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  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidKnown type III effector protein(s) expressed or relevant characteristic(s)Reference(s) or source
    P. aeruginosa strains
     PA103ExoT, ExoUB. H. Iglewski
     PA103ΔexoUExoT4
     PA103exoT::TcExoU3
     PA103ΔexoUexoT::TcNoneThis study
     PA103toxA::ΩExoT, ExoUB. H. Iglewski
     PA103exsA::ΩNone7
    Plasmids
     pMOBexoT::TcAllelic replacement vector encoding tetracycline-interruptedexoT3, 18
     pUCP18pUC-derived cloning vector able to replicate in P. aeruginosa17
     pUCPexoSEncodes the wild-type ADP-ribosyltransferase ExoS (100% activity)12
     pUCPexoSE381AEncodes the noncatalytic mutant ExoSE381A (0.02% activity)This study and 13
     pUCPexoTEncodes the ADP-ribosyltransferase ExoT (0.2% activity)21
     pUCPexoYEncodes the adenylate cyclase ExoY23
     pUCPexoYK81MEncodes the noncatalytic mutant ExoYK81M23
  • Table 2.

    Biological effects of the various bacterial strains on CHO cells

    StrainEffector(s)% Altered morphologya% Trypan blue stainedaDecreased viabilityb
    None (uninfected)6 ± 20NAc
    PA103ExoT, ExoU53 ± 253 ± 2+
    PA103exoT::TcExoU46 ± 846 ± 8+
    PA103toxA::ΩdExoT, ExoUNDeND+
    PA103ΔexoUExoT98 ± 20−
    PA103ΔexoUexoT::TcNone6 ± 20−
    PA103exsA::ΩNoneNCf0−
    PA103ΔexoUexoT::Tc pUCPNone 50−
    PA103ΔexoUexoT::Tc pUCPexoSExoS90 ± 30−
    PA103ΔexoUexoT::Tc pUCPexoSE381AExoSE381A99 ± 10−
    PA103ΔexoUexoT::Tc pUCPexoTExoT990−
    PA103ΔexoUexoT::Tc pUCPexoYExoY95 ± 60−
    PA103ΔexoUexoT::Tc pUCPexoYK81MExoYK81M7 ± 40−
    • ↵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

    ProteinEnzymatic activityEffect on CHO cells
    ExoSADP-ribosyltransferaseMorphological alterations
    ExoTADP-ribosyltransferaseMorphological alterations
    ExoUUnknownAcute cytotoxicity: membrane permeability, loss of viability
    Morphological alterations
    ExoYAdenylate cyclaseMorphological alterations
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Biological Effects of Pseudomonas aeruginosa Type III-Secreted Proteins on CHO Cells
Amy J. Vallis, Viviane Finck-Barbançon, Timothy L. Yahr, Dara W. Frank
Infection and Immunity Apr 1999, 67 (4) 2040-2044; DOI: 10.1128/IAI.67.4.2040-2044.1999

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Biological Effects of Pseudomonas aeruginosa Type III-Secreted Proteins on CHO Cells
Amy J. Vallis, Viviane Finck-Barbançon, Timothy L. Yahr, Dara W. Frank
Infection and Immunity Apr 1999, 67 (4) 2040-2044; DOI: 10.1128/IAI.67.4.2040-2044.1999
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KEYWORDS

bacterial toxins
Exotoxins
Pseudomonas aeruginosa

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