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Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

Zrieq R, Sana TG, Vergin S, Garvis S, Volfson I, Bleves S, Voulhoux R, Hegemann JH - Front Cell Infect Microbiol (2015)

Bottom Line: Fifty-one candidates were selected in athree-round screening process.By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa.Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

View Article: PubMed Central - PubMed

Affiliation: Institut für Funktionelle Genomforschung der Mikroorganismen, Heinrich-Heine-Universität Düsseldorf Düsseldorf, Germany.

ABSTRACT
Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

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Expression patterns of GFP-Pec3 in HEp-2 transfected cells. HEp-2 cells were transfected with a human expression vector carrying GFP or GFP-Pec3 for 18 h. Cells were then fixed and stained with DAPI (blue) to visualize nuclei and with rhodamine-phalloidin (red) to visualize F-actin.
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Figure 6: Expression patterns of GFP-Pec3 in HEp-2 transfected cells. HEp-2 cells were transfected with a human expression vector carrying GFP or GFP-Pec3 for 18 h. Cells were then fixed and stained with DAPI (blue) to visualize nuclei and with rhodamine-phalloidin (red) to visualize F-actin.

Mentions: Ectopic production of GFP-Pec3 in epithelial cells showed a diffuse GFP pattern similar to GFP expressing control cells indicating no particular localization of this effector (Figure 6). A similar localization pattern was also observed in yeast (data not shown). These results suggest that the target of Pec3 cannot be identified by localization studies.


Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

Zrieq R, Sana TG, Vergin S, Garvis S, Volfson I, Bleves S, Voulhoux R, Hegemann JH - Front Cell Infect Microbiol (2015)

Expression patterns of GFP-Pec3 in HEp-2 transfected cells. HEp-2 cells were transfected with a human expression vector carrying GFP or GFP-Pec3 for 18 h. Cells were then fixed and stained with DAPI (blue) to visualize nuclei and with rhodamine-phalloidin (red) to visualize F-actin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644809&req=5

Figure 6: Expression patterns of GFP-Pec3 in HEp-2 transfected cells. HEp-2 cells were transfected with a human expression vector carrying GFP or GFP-Pec3 for 18 h. Cells were then fixed and stained with DAPI (blue) to visualize nuclei and with rhodamine-phalloidin (red) to visualize F-actin.
Mentions: Ectopic production of GFP-Pec3 in epithelial cells showed a diffuse GFP pattern similar to GFP expressing control cells indicating no particular localization of this effector (Figure 6). A similar localization pattern was also observed in yeast (data not shown). These results suggest that the target of Pec3 cannot be identified by localization studies.

Bottom Line: Fifty-one candidates were selected in athree-round screening process.By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa.Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

View Article: PubMed Central - PubMed

Affiliation: Institut für Funktionelle Genomforschung der Mikroorganismen, Heinrich-Heine-Universität Düsseldorf Düsseldorf, Germany.

ABSTRACT
Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

Show MeSH
Related in: MedlinePlus