Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells - PubMed (original) (raw)

Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells

A J Vallis et al. Infect Immun. 1999 Apr.

Abstract

A strain of Pseudomonas aeruginosa that fails to express known type III-secreted effector proteins was constructed as an expression host. Individual effectors were expressed in trans, and their biological effects on CHO cells were assessed in an acute cellular infection model. Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology. As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.

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Figures

FIG. 1

Extracellular protein profiles, Western blot analysis, and infection of CHO cells with parental (PA103) and mutant (PA103Δ_exoU_, PA103_exoT_::Tc, and PA103Δ_exoUexoT_::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), PA103Δ_exoU_ (lanes 3 and 4), PA103_exoT_::Tc (lanes 5 and 6), and PA103Δ_exoUexoT_::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 PA103_exoT_::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 PA103Δ_exoUexoT_::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 PA103Δ_exoUexoT_::Tc pUCP18 (lane 1), PA103Δ_exoUexoT_::Tc pUCP_exoS_ (lane 2), PA103Δ_exoUexoT_::Tc pUCP_exoS_E381A (lane 3), PA103Δ_exoUexoT_::Tc pUCP_exoT_ (lane 4), PA103Δ_exoUexoT_::Tc pUCP_exoY_ (lane 5), and PA103Δ_exoUexoT_::Tc pUCP_exoY_K81M (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 PA103Δ_exoUexoT_::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 PA103Δ_exoUexoT_::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 PA103Δ_exoUexoT_::Tc pUCP18, PA103Δ_exoUexoT_::Tc pUCP_exoS_, PA103Δ_exoUexoT_::Tc pUCP_exoS_E381A, or PA103_exoUexoT_::Tc pUCP_exoT_. Activity is normalized to CFU and expressed as 10−4 femtomoles of ADPRT (femtomoles of ADP-ribose transferred to soybean trypsin inhibitor).

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Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells - PubMed (original) (raw)