Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S

Abstract

Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa 388 and an isogenic mutant strain, 388DeltaexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388DeltaexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388DeltaexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.

FIG. 1

Mobility shift of Ras in HT29 cell lysates modified by ExoS in vitro and in vivo. (A) Ras modification in vitro by purified ExoS. HT29 cell monolayers were radiolabeled with [³⁵S]methionine for 18 h and then lysed, and extracts were incubated for 30 min in the presence of buffer (−ExoS) or purified recombinant ExoS (+ExoS). Ras was then immunoprecipitated with monoclonal antibody Y13-259 coupled to anti-rat IgG plus protein A-Sepharose. Proteins were separated by SDS-PAGE and visualized by fluorography. M, modified Ras; U, unmodified Ras. (B) Ras modification in vivo by ExoS-producing bacteria. [³⁵S]methionine-labeled HT29 cells were incubated for 3 h with McCoy’s-BSA alone (0) or with 10⁸ CFU of ExoS-producing (388) or non-ExoS-producing (ΔS) bacterial strains as indicated. Bacteria were removed, cells were lysed, and Ras was immunoprecipitated and detected as described for panel A. Molecular masses (in kilodaltons) are indicated.

FIG. 2

Comparison of Ras modification by strain 388 and by ExoS secreted into the medium. (A) Lack of Ras modification by extracellularly secreted ExoS. Unlabeled HT29 cells were incubated for 3 h with medium alone (0) or with 10⁸ CFU of strain 388. The 388 coculture medium was then removed, filtered, and applied to a fresh HT29 cell monolayer for 3 h (0+S). Ras was immunoprecipitated from lysates of cells cultured with strain 388 bacteria or with secreted ExoS. The electrophoretic mobility of Ras was evaluated following SDS-PAGE, immunoblotting with mouse monoclonal anti-Ras, and detection by ECL. M, modified Ras; U, unmodified Ras. (B) Lack of Ras modification by secreted ExoS in the presence of ΔS bacteria. HT29 cells were incubated for 3 h with 10⁸ CFU of strain 388, ΔS alone, or ΔS in the presence of secreted ExoS prepared as described for panel A (ΔS + S). The electrophoretic mobility of Ras was examined as described for panel A. Molecular masses (in kilodaltons) are indicated.

FIG. 3

Comparison of Ras modification in HT29 cells by different P. aeruginosa strains. (A) HT29 cells were incubated with 10⁸ CFU of strain 388, PAK, DG1, or PA103 (103), as indicated; then Ras was immunoprecipitated and detected by immunoblotting as described for Fig. 2A. M, modified Ras; U, unmodified Ras. (B) HT29 cells were incubated with 10⁸ CFU of strain 388 or ΔT and analyzed as described above. Molecular masses (in kilodaltons) are indicated.

FIG. 4

Dependence of in vivo Ras modification on time of exposure to bacteria and bacterial concentration. (A) Time required for Ras modification. HT29 cells were incubated with no bacteria (0) or with 10⁸ CFU of strain 388 bacteria for 1, 2, or 3 h, as indicated. Ras was immunoprecipitated from cell lysates and detected by SDS-PAGE and immunoblotting. M, modified Ras; U, unmodified Ras. (B) Bacterial concentration required for Ras modification. Cells were incubated for 3 h with 0 to 10⁸ CFU of strain 388 bacteria, as indicated, and Ras modification was examined as for panel A. Molecular masses (in kilodaltons) are indicated.

FIG. 5

Correlation of Ras modification with reduced DNA synthesis. DNA synthesis was measured in HT29 cells which had been seeded in 48-well plates at 10⁵ cells/well, grown for 48 h, then incubated with 0 to 10⁸ CFU of strain 388 or ΔS/ml in McCoy’s-BSA for 3 h. At this time, monolayers were washed to remove bacteria, and McCoy’s-FBS-GC containing 1 μCi of [³H]thymidine/ml was added. DNA synthesis was determined after 18 h and is expressed as percent reduction in [³H]thymidine uptake compared to that in nonbacterially treated controls. Results are expressed as means and standard deviations of a single assay performed in quadruplicate and are representative of two independent studies. Percent reduction in DNA synthesis was compared to percent modification of Ras by incubating HT29 cells in an identical manner with increasing concentrations of strain 388 bacteria. The percentage of immunoprecipitated Ras modified was determined by densitometric analysis of a representative image.

FIG. 6

Investigation of Rap1 modification. (A) Examination of Rap1 mobility in HT29 cells following exposure to 388 or ΔS bacteria. [³⁵S]methionine-labeled cells were prepared and cocultured with the indicated bacterial strains as described for Fig. 1B. Rap1 proteins were immunoprecipitated with rabbit polyclonal Rap1–Krev-1 (121) antibody and detected by SDS-PAGE, followed by fluorography. Arrows indicate 20- and 24-kDa Rap1 proteins. (B) Comparison of ADP-ribosylation of Ras and Rap1 in HT29 cells exposed to strain 388. Cellular proteins were labeled with [³⁵S]methionine (³⁵S), or intracellular NAD pools were labeled by treating HT29 monolayers with 5 μg of actinomycin D/ml to reduce RNA synthesis and then radiolabeling with [³H]adenosine (³H) for 18 h. Cells were either left untreated (0) or exposed to 10⁸ CFU of strain 388 bacteria/ml. Ras and Rap1 were immunoprecipitated and subjected to SDS-PAGE and fluorography as described above. M, modified Ras; U, unmodified Ras. Molecular masses (in kilodaltons) are indicated.