Limits...
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.

Show MeSH

Related in: MedlinePlus

Yeast growth phenotype induced by expression of selected pec genes in yeast. (A) Serial dilution patch test of yeast cells carrying the empty vector (control) or the 3 selected candidates obtained from the screen (Pec1, Pec2, and Pec3). Cells were dropped on selective non-inducing medium (Glucose) or selective induced medium (Galactose) as indicated. (B) Serial dilution patch test of yeast cells carrying the empty plasmid (control) or full length genes of the 3 selected candidates (Pec1, Pec2, and Pec3). Cells were dropped on selective induced medium (Galactose) as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4644809&req=5

Figure 3: Yeast growth phenotype induced by expression of selected pec genes in yeast. (A) Serial dilution patch test of yeast cells carrying the empty vector (control) or the 3 selected candidates obtained from the screen (Pec1, Pec2, and Pec3). Cells were dropped on selective non-inducing medium (Glucose) or selective induced medium (Galactose) as indicated. (B) Serial dilution patch test of yeast cells carrying the empty plasmid (control) or full length genes of the 3 selected candidates (Pec1, Pec2, and Pec3). Cells were dropped on selective induced medium (Galactose) as indicated.

Mentions: Expression of pec1,pec2, and pec3 from the original screen plasmids resulted in impaired yeast growth (Figure 3A). In order to test whether the expression of full length pec genes also would impair yeast growth, we produced the full length Pec1, Pec2, and Pec3 proteins in yeast and tested them by serial dilution patch tests (Figure 3B). In comparison to the yeast growth phenotypes induced by the initial screen plasmids, the production of full length Pecs showed a significant but attenuated yeast growth defect phenotype, as detectable by smaller and less numerous colonies. These differences in growth are likely due to alterations in protein production levels, protein folding and/or protein modification. We checked by sequencing that the differences in growth of yeast cells expressing full length and genomic fragment of Pec1 and Pec2 were not due to enhanced translation given by additional genes or small RNAs within the cloned fragments (data not shown). We therefore concluded that expression of the 3 full-length pec genes validated the original yeast growth phenotype, supporting their possible role as P. aeruginosa virulence factors.


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)

Yeast growth phenotype induced by expression of selected pec genes in yeast. (A) Serial dilution patch test of yeast cells carrying the empty vector (control) or the 3 selected candidates obtained from the screen (Pec1, Pec2, and Pec3). Cells were dropped on selective non-inducing medium (Glucose) or selective induced medium (Galactose) as indicated. (B) Serial dilution patch test of yeast cells carrying the empty plasmid (control) or full length genes of the 3 selected candidates (Pec1, Pec2, and Pec3). Cells were dropped on selective induced medium (Galactose) as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Yeast growth phenotype induced by expression of selected pec genes in yeast. (A) Serial dilution patch test of yeast cells carrying the empty vector (control) or the 3 selected candidates obtained from the screen (Pec1, Pec2, and Pec3). Cells were dropped on selective non-inducing medium (Glucose) or selective induced medium (Galactose) as indicated. (B) Serial dilution patch test of yeast cells carrying the empty plasmid (control) or full length genes of the 3 selected candidates (Pec1, Pec2, and Pec3). Cells were dropped on selective induced medium (Galactose) as indicated.
Mentions: Expression of pec1,pec2, and pec3 from the original screen plasmids resulted in impaired yeast growth (Figure 3A). In order to test whether the expression of full length pec genes also would impair yeast growth, we produced the full length Pec1, Pec2, and Pec3 proteins in yeast and tested them by serial dilution patch tests (Figure 3B). In comparison to the yeast growth phenotypes induced by the initial screen plasmids, the production of full length Pecs showed a significant but attenuated yeast growth defect phenotype, as detectable by smaller and less numerous colonies. These differences in growth are likely due to alterations in protein production levels, protein folding and/or protein modification. We checked by sequencing that the differences in growth of yeast cells expressing full length and genomic fragment of Pec1 and Pec2 were not due to enhanced translation given by additional genes or small RNAs within the cloned fragments (data not shown). We therefore concluded that expression of the 3 full-length pec genes validated the original yeast growth phenotype, supporting their possible role as P. aeruginosa virulence factors.

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