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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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IpaJ Secretion and Translocation Are Dependent on an Intact Type III Secretion System(A) Wild-type and BS547 (mxiM1) [50] Shigella, a TTSS-defective strain, that carry a low-copy number plasmid that expresses IpaJ-FLAG from its endogenous promotor were grown in the presence of Congo Red, an inducer of the Shigella TTSS. Proteins from culture supernatants and whole-cells were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody.(B) HeLa cells were infected with either wild-type or BS547 (mxiM1) Shigella that express IpaJ-FLAG at an MOI of 100:1. One hour after infection, the cells were lysed and fractionated into soluble and insoluble fractions. Proteins from each fraction were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody. Translocated proteins are found in the soluble fraction, while those from unlysed bacteria are found in the insoluble fraction.
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ppat-0040009-g005: IpaJ Secretion and Translocation Are Dependent on an Intact Type III Secretion System(A) Wild-type and BS547 (mxiM1) [50] Shigella, a TTSS-defective strain, that carry a low-copy number plasmid that expresses IpaJ-FLAG from its endogenous promotor were grown in the presence of Congo Red, an inducer of the Shigella TTSS. Proteins from culture supernatants and whole-cells were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody.(B) HeLa cells were infected with either wild-type or BS547 (mxiM1) Shigella that express IpaJ-FLAG at an MOI of 100:1. One hour after infection, the cells were lysed and fractionated into soluble and insoluble fractions. Proteins from each fraction were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody. Translocated proteins are found in the soluble fraction, while those from unlysed bacteria are found in the insoluble fraction.

Mentions: Of the three proteins of unknown function we expressed in yeast, only one, IpaJ, resulted in yeast growth inhibition. To determine if we had identified a new substrate of the Shigella TTSS, a plasmid that encodes IpaJ fused to 3x FLAG tag was introduced into wild type Shigella strain as well as a Shigella strain defective in TTSS due to a mutation in mxiM1 [50], a component of the secretion apparatus. As demonstrated in Figure 5A, IpaJ is only detected by western blot analyses in the supernatants of wild-type Shigella after the addition of Congo red, a TTSS inducer, to the media [51]. Similarly, as shown in Figure 5B, detectable levels of IpaJ within host cells during the course of an infection is also dependent on an intact TTSS. Secretion of IpaJ appeared to be ∼100 times lower than that of either IpgB1 or OspF (data not shown). This difference in secretion levels may explain the lack of detection of IpaJ in prior analyses of native proteins in supernatant preparations of Shigella mutants that constitutively secrete effector proteins [16]. Thus, our growth inhibition screen led to the identification of a new effector protein that had been previously missed by other experimental approaches.


A functional genomic yeast screen to identify pathogenic bacterial proteins.

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

IpaJ Secretion and Translocation Are Dependent on an Intact Type III Secretion System(A) Wild-type and BS547 (mxiM1) [50] Shigella, a TTSS-defective strain, that carry a low-copy number plasmid that expresses IpaJ-FLAG from its endogenous promotor were grown in the presence of Congo Red, an inducer of the Shigella TTSS. Proteins from culture supernatants and whole-cells were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody.(B) HeLa cells were infected with either wild-type or BS547 (mxiM1) Shigella that express IpaJ-FLAG at an MOI of 100:1. One hour after infection, the cells were lysed and fractionated into soluble and insoluble fractions. Proteins from each fraction were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody. Translocated proteins are found in the soluble fraction, while those from unlysed bacteria are found in the insoluble fraction.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040009-g005: IpaJ Secretion and Translocation Are Dependent on an Intact Type III Secretion System(A) Wild-type and BS547 (mxiM1) [50] Shigella, a TTSS-defective strain, that carry a low-copy number plasmid that expresses IpaJ-FLAG from its endogenous promotor were grown in the presence of Congo Red, an inducer of the Shigella TTSS. Proteins from culture supernatants and whole-cells were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody.(B) HeLa cells were infected with either wild-type or BS547 (mxiM1) Shigella that express IpaJ-FLAG at an MOI of 100:1. One hour after infection, the cells were lysed and fractionated into soluble and insoluble fractions. Proteins from each fraction were separated by SDS-PAGE and subjected to western blot analyses with anti-FLAG antibody. Translocated proteins are found in the soluble fraction, while those from unlysed bacteria are found in the insoluble fraction.
Mentions: Of the three proteins of unknown function we expressed in yeast, only one, IpaJ, resulted in yeast growth inhibition. To determine if we had identified a new substrate of the Shigella TTSS, a plasmid that encodes IpaJ fused to 3x FLAG tag was introduced into wild type Shigella strain as well as a Shigella strain defective in TTSS due to a mutation in mxiM1 [50], a component of the secretion apparatus. As demonstrated in Figure 5A, IpaJ is only detected by western blot analyses in the supernatants of wild-type Shigella after the addition of Congo red, a TTSS inducer, to the media [51]. Similarly, as shown in Figure 5B, detectable levels of IpaJ within host cells during the course of an infection is also dependent on an intact TTSS. Secretion of IpaJ appeared to be ∼100 times lower than that of either IpgB1 or OspF (data not shown). This difference in secretion levels may explain the lack of detection of IpaJ in prior analyses of native proteins in supernatant preparations of Shigella mutants that constitutively secrete effector proteins [16]. Thus, our growth inhibition screen led to the identification of a new effector protein that had been previously missed by other experimental approaches.

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