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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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The Addition of Stressors Elicits Additional Yeast Growth Inhibition Phenotypes by Translocated ProteinsThe heatmap summarizes the relative growth at t = 48 h of yeast that express the Shigella GFP fusion proteins or GFP grown under each of five conditions: no stressor, 6 mM caffeine, 3 μg/ml nocodazole, 0.5 M sorbitol, and 0.5M NaCl. Each cell represents the mean OD600 measurements at 48 h of six independent yeast colonies expressing the same bacterial protein from a low-copy yeast expression plasmid grown under the same condition. Rows and columns were clustered by the complete linkage algorithm using the standard Euclidean distance metric operating directly on data vectors (i.e., without normalization). The proteins labeled in red are translocated, those labeled in black are non-translocated, and the unknowns in blue. GFP control is labeled in gray.
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ppat-0040009-g003: The Addition of Stressors Elicits Additional Yeast Growth Inhibition Phenotypes by Translocated ProteinsThe heatmap summarizes the relative growth at t = 48 h of yeast that express the Shigella GFP fusion proteins or GFP grown under each of five conditions: no stressor, 6 mM caffeine, 3 μg/ml nocodazole, 0.5 M sorbitol, and 0.5M NaCl. Each cell represents the mean OD600 measurements at 48 h of six independent yeast colonies expressing the same bacterial protein from a low-copy yeast expression plasmid grown under the same condition. Rows and columns were clustered by the complete linkage algorithm using the standard Euclidean distance metric operating directly on data vectors (i.e., without normalization). The proteins labeled in red are translocated, those labeled in black are non-translocated, and the unknowns in blue. GFP control is labeled in gray.

Mentions: The addition of the stressors to the media elicited marked changes in the phenotypes of two translocated proteins (Figure 3). OspB weakly inhibited growth in standard conditions, but in the presence of caffeine low-level OspB expression severely inhibited yeast growth. The phenotype of IcsA was similarly amplified by the addition of nocodazole. Although these were the only strictly conditional phenotypes, yeast expressing proteins that conferred intermediate growth phenotypes when expressed at low-levels (IpgB1, OspC1, OspD3, OspF and ParA) displayed different sensitivities to the four stressors, implying that the phenotypes conferred by each of these proteins is due to a specific activity of the protein. In summary, the stress conditions increased the sensitivity of the assay with no loss of specificity.


A functional genomic yeast screen to identify pathogenic bacterial proteins.

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

The Addition of Stressors Elicits Additional Yeast Growth Inhibition Phenotypes by Translocated ProteinsThe heatmap summarizes the relative growth at t = 48 h of yeast that express the Shigella GFP fusion proteins or GFP grown under each of five conditions: no stressor, 6 mM caffeine, 3 μg/ml nocodazole, 0.5 M sorbitol, and 0.5M NaCl. Each cell represents the mean OD600 measurements at 48 h of six independent yeast colonies expressing the same bacterial protein from a low-copy yeast expression plasmid grown under the same condition. Rows and columns were clustered by the complete linkage algorithm using the standard Euclidean distance metric operating directly on data vectors (i.e., without normalization). The proteins labeled in red are translocated, those labeled in black are non-translocated, and the unknowns in blue. GFP control is labeled in gray.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040009-g003: The Addition of Stressors Elicits Additional Yeast Growth Inhibition Phenotypes by Translocated ProteinsThe heatmap summarizes the relative growth at t = 48 h of yeast that express the Shigella GFP fusion proteins or GFP grown under each of five conditions: no stressor, 6 mM caffeine, 3 μg/ml nocodazole, 0.5 M sorbitol, and 0.5M NaCl. Each cell represents the mean OD600 measurements at 48 h of six independent yeast colonies expressing the same bacterial protein from a low-copy yeast expression plasmid grown under the same condition. Rows and columns were clustered by the complete linkage algorithm using the standard Euclidean distance metric operating directly on data vectors (i.e., without normalization). The proteins labeled in red are translocated, those labeled in black are non-translocated, and the unknowns in blue. GFP control is labeled in gray.
Mentions: The addition of the stressors to the media elicited marked changes in the phenotypes of two translocated proteins (Figure 3). OspB weakly inhibited growth in standard conditions, but in the presence of caffeine low-level OspB expression severely inhibited yeast growth. The phenotype of IcsA was similarly amplified by the addition of nocodazole. Although these were the only strictly conditional phenotypes, yeast expressing proteins that conferred intermediate growth phenotypes when expressed at low-levels (IpgB1, OspC1, OspD3, OspF and ParA) displayed different sensitivities to the four stressors, implying that the phenotypes conferred by each of these proteins is due to a specific activity of the protein. In summary, the stress conditions increased the sensitivity of the assay with no loss of specificity.

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