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A functional genomic yeast screen to identify pathogenic bacterial proteins.

Slagowski NL, Kramer RW, Morrison MF, LaBaer J, Lesser CF - PLoS Pathog. (2008)

Bottom Line: This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods.In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens.In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts, United States of America.

ABSTRACT
Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.

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Related in: MedlinePlus

Growth Phenotypes Conferred by Expression of Shigella Proteins in YeastEach box-and-whisker plot summarizes the OD600 measurements of 22 independent yeast cultures expressing a single Shigella protein at t = 48 h. Proteins shown in yellow represent those whose expression resulted in a significant reduction in growth when compared to the control (p < 0.001 in a Wilcoxon two-sample test with Bonferroni correction). “Control” refers to yeast that carry empty vector. The boxes enclose approximately one quartile either side of the median. The whiskers delimit the ∼95% confidence interval for the mean (using default rendering parameters in the statistical computing software package R [55]).
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ppat-0040009-g004: Growth Phenotypes Conferred by Expression of Shigella Proteins in YeastEach box-and-whisker plot summarizes the OD600 measurements of 22 independent yeast cultures expressing a single Shigella protein at t = 48 h. Proteins shown in yellow represent those whose expression resulted in a significant reduction in growth when compared to the control (p < 0.001 in a Wilcoxon two-sample test with Bonferroni correction). “Control” refers to yeast that carry empty vector. The boxes enclose approximately one quartile either side of the median. The whiskers delimit the ∼95% confidence interval for the mean (using default rendering parameters in the statistical computing software package R [55]).

Mentions: Another explanation for the lack of detection of yeast growth inhibition due to expression of bacterial proteins is that fusion to GFP could potentially interfere with the activity and/or folding of the proteins and thus mask phenotypes. To address this possibility we conducted growth assays on yeast that express the Shigella proteins in the absence of GFP. The absence of GFP decreased the overall toxicity of almost all the Shigella proteins when expressed from a high-copy number plasmid (Figure 4 vs. Figure 1). High-level expression of all the Shigella GFP fusion proteins inhibited growth more often than their non-GFP counterparts (75% vs. 38%).


A functional genomic yeast screen to identify pathogenic bacterial proteins.

Slagowski NL, Kramer RW, Morrison MF, LaBaer J, Lesser CF - PLoS Pathog. (2008)

Growth Phenotypes Conferred by Expression of Shigella Proteins in YeastEach box-and-whisker plot summarizes the OD600 measurements of 22 independent yeast cultures expressing a single Shigella protein at t = 48 h. Proteins shown in yellow represent those whose expression resulted in a significant reduction in growth when compared to the control (p < 0.001 in a Wilcoxon two-sample test with Bonferroni correction). “Control” refers to yeast that carry empty vector. The boxes enclose approximately one quartile either side of the median. The whiskers delimit the ∼95% confidence interval for the mean (using default rendering parameters in the statistical computing software package R [55]).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211553&req=5

ppat-0040009-g004: Growth Phenotypes Conferred by Expression of Shigella Proteins in YeastEach box-and-whisker plot summarizes the OD600 measurements of 22 independent yeast cultures expressing a single Shigella protein at t = 48 h. Proteins shown in yellow represent those whose expression resulted in a significant reduction in growth when compared to the control (p < 0.001 in a Wilcoxon two-sample test with Bonferroni correction). “Control” refers to yeast that carry empty vector. The boxes enclose approximately one quartile either side of the median. The whiskers delimit the ∼95% confidence interval for the mean (using default rendering parameters in the statistical computing software package R [55]).
Mentions: Another explanation for the lack of detection of yeast growth inhibition due to expression of bacterial proteins is that fusion to GFP could potentially interfere with the activity and/or folding of the proteins and thus mask phenotypes. To address this possibility we conducted growth assays on yeast that express the Shigella proteins in the absence of GFP. The absence of GFP decreased the overall toxicity of almost all the Shigella proteins when expressed from a high-copy number plasmid (Figure 4 vs. Figure 1). High-level expression of all the Shigella GFP fusion proteins inhibited growth more often than their non-GFP counterparts (75% vs. 38%).

Bottom Line: This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods.In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens.In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts, United States of America.

ABSTRACT
Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.

Show MeSH
Related in: MedlinePlus