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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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Scheme of the genome wide screen of P. aeruginosa PA14 in yeast. The correlation between amount of library DNA and genome fold was determined by transforming yeast cells with serial dilutions of the library DNA and counting the resulting yeast transformants. Having the correlation established, 1.14 μg of the library DNA (representing 3 times coverage of the PA14 genome) was transformed into yeast cells (A). For the primary screen, transformed yeast cells were selected on a synthetic defined medium lacking histidine (SD-HIS) supplemented with raffinose (B). Yeast colonies obtained ~1.3 105 transformants, representing approximately 2.3 times of the PA14 genome) were transferred by replica platting to a solid SD-HIS containing galactose and Phloxine B medium (C), arrows and numbers (1, 2, and 3) in (C) represent examples for growth phenotypes observed (non-growing, dead phloxine-positive and smaller colonies, respectively), see also Figure S1. Colonies showed growth phenotype (1186 candidates) were streaked out on plates (SD-HIS) supplemented with raffinose (D). For the secondary screen, independent candidates from (D) were subjected to serial dilution patch test and positive Pseudomonas effector candidates were selected (E). Plasmids were then isolated (F) for the tertiary screen and differentiated by restriction analysis (G). Plasmids were retransformed into yeast (H) and transformants from each candidate were subjected for serial dilution patch test. Fifty four candidates showed growth phenotype (I).
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Figure 1: Scheme of the genome wide screen of P. aeruginosa PA14 in yeast. The correlation between amount of library DNA and genome fold was determined by transforming yeast cells with serial dilutions of the library DNA and counting the resulting yeast transformants. Having the correlation established, 1.14 μg of the library DNA (representing 3 times coverage of the PA14 genome) was transformed into yeast cells (A). For the primary screen, transformed yeast cells were selected on a synthetic defined medium lacking histidine (SD-HIS) supplemented with raffinose (B). Yeast colonies obtained ~1.3 105 transformants, representing approximately 2.3 times of the PA14 genome) were transferred by replica platting to a solid SD-HIS containing galactose and Phloxine B medium (C), arrows and numbers (1, 2, and 3) in (C) represent examples for growth phenotypes observed (non-growing, dead phloxine-positive and smaller colonies, respectively), see also Figure S1. Colonies showed growth phenotype (1186 candidates) were streaked out on plates (SD-HIS) supplemented with raffinose (D). For the secondary screen, independent candidates from (D) were subjected to serial dilution patch test and positive Pseudomonas effector candidates were selected (E). Plasmids were then isolated (F) for the tertiary screen and differentiated by restriction analysis (G). Plasmids were retransformed into yeast (H) and transformants from each candidate were subjected for serial dilution patch test. Fifty four candidates showed growth phenotype (I).

Mentions: To identify bacterial proteins that perturb essential and conserved eukaryotic processes, we constructed a genomic library of the P.aeruginosa PA14 strain and performed a three steps genome-wide screen in yeast for bacterial genomic fragments inducing a yeast growth phenotype. After transforming the library into yeast and platted onto non-inducing media agar plates (Figure 1A), approximately 1.3 × 105 yeast colonies were obtained indicating that at least the equivalent of 2.3-fold of the P. aeruginosa genome was actually transformed (Figure 1B). The screen was then processed in 3 steps. In the first step (Figures 1C,D), we performed a primary negative selection by transferring yeast colonies via replica platting onto media agar plates containing galactose and Phloxin B (Figure 1C). We identified and isolated 1186 colonies that did not grow, displayed smaller size and/or increased pink staining on inducing media plates (Figures 1C,D and Figure S1). In the secondary screen, performed by a serial dilution patch test, we selected among the 1186 primary candidates, 257 secondary candidates showing a growth arrest phenotype (Figure 1E). Restriction analysis of plasmid DNA from the 257 yeast candidates revealed that 38 yeast candidates harbored two different plasmids that carried two different inserts, resulting in a total of 295 plasmids (Figures 1F,G). Finally, in the tertiary screen, among the 295 plasmids retransformed in yeast (Figure 1H), 54 potential P. aeruginosa PA14 genomic fragment carrying-plasmids showed a growth phenotype defect by serial dilution patch test (Figure 1I). All plasmid inserts were sequenced and corresponding genes were subjected to further analysis.


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)

Scheme of the genome wide screen of P. aeruginosa PA14 in yeast. The correlation between amount of library DNA and genome fold was determined by transforming yeast cells with serial dilutions of the library DNA and counting the resulting yeast transformants. Having the correlation established, 1.14 μg of the library DNA (representing 3 times coverage of the PA14 genome) was transformed into yeast cells (A). For the primary screen, transformed yeast cells were selected on a synthetic defined medium lacking histidine (SD-HIS) supplemented with raffinose (B). Yeast colonies obtained ~1.3 105 transformants, representing approximately 2.3 times of the PA14 genome) were transferred by replica platting to a solid SD-HIS containing galactose and Phloxine B medium (C), arrows and numbers (1, 2, and 3) in (C) represent examples for growth phenotypes observed (non-growing, dead phloxine-positive and smaller colonies, respectively), see also Figure S1. Colonies showed growth phenotype (1186 candidates) were streaked out on plates (SD-HIS) supplemented with raffinose (D). For the secondary screen, independent candidates from (D) were subjected to serial dilution patch test and positive Pseudomonas effector candidates were selected (E). Plasmids were then isolated (F) for the tertiary screen and differentiated by restriction analysis (G). Plasmids were retransformed into yeast (H) and transformants from each candidate were subjected for serial dilution patch test. Fifty four candidates showed growth phenotype (I).
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Figure 1: Scheme of the genome wide screen of P. aeruginosa PA14 in yeast. The correlation between amount of library DNA and genome fold was determined by transforming yeast cells with serial dilutions of the library DNA and counting the resulting yeast transformants. Having the correlation established, 1.14 μg of the library DNA (representing 3 times coverage of the PA14 genome) was transformed into yeast cells (A). For the primary screen, transformed yeast cells were selected on a synthetic defined medium lacking histidine (SD-HIS) supplemented with raffinose (B). Yeast colonies obtained ~1.3 105 transformants, representing approximately 2.3 times of the PA14 genome) were transferred by replica platting to a solid SD-HIS containing galactose and Phloxine B medium (C), arrows and numbers (1, 2, and 3) in (C) represent examples for growth phenotypes observed (non-growing, dead phloxine-positive and smaller colonies, respectively), see also Figure S1. Colonies showed growth phenotype (1186 candidates) were streaked out on plates (SD-HIS) supplemented with raffinose (D). For the secondary screen, independent candidates from (D) were subjected to serial dilution patch test and positive Pseudomonas effector candidates were selected (E). Plasmids were then isolated (F) for the tertiary screen and differentiated by restriction analysis (G). Plasmids were retransformed into yeast (H) and transformants from each candidate were subjected for serial dilution patch test. Fifty four candidates showed growth phenotype (I).
Mentions: To identify bacterial proteins that perturb essential and conserved eukaryotic processes, we constructed a genomic library of the P.aeruginosa PA14 strain and performed a three steps genome-wide screen in yeast for bacterial genomic fragments inducing a yeast growth phenotype. After transforming the library into yeast and platted onto non-inducing media agar plates (Figure 1A), approximately 1.3 × 105 yeast colonies were obtained indicating that at least the equivalent of 2.3-fold of the P. aeruginosa genome was actually transformed (Figure 1B). The screen was then processed in 3 steps. In the first step (Figures 1C,D), we performed a primary negative selection by transferring yeast colonies via replica platting onto media agar plates containing galactose and Phloxin B (Figure 1C). We identified and isolated 1186 colonies that did not grow, displayed smaller size and/or increased pink staining on inducing media plates (Figures 1C,D and Figure S1). In the secondary screen, performed by a serial dilution patch test, we selected among the 1186 primary candidates, 257 secondary candidates showing a growth arrest phenotype (Figure 1E). Restriction analysis of plasmid DNA from the 257 yeast candidates revealed that 38 yeast candidates harbored two different plasmids that carried two different inserts, resulting in a total of 295 plasmids (Figures 1F,G). Finally, in the tertiary screen, among the 295 plasmids retransformed in yeast (Figure 1H), 54 potential P. aeruginosa PA14 genomic fragment carrying-plasmids showed a growth phenotype defect by serial dilution patch test (Figure 1I). All plasmid inserts were sequenced and corresponding genes were subjected to further analysis.

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