Proteomic identification of novel secreted anti-bacterial toxins of the Serratia marcescens Type VI secretion system

Maximilian J. Fritsch, Katharina Trunk, Juliana Alcoforado Diniz, Manman Guo, Matthias Trost (Lead / Corresponding author), Sarah Jane Coulthurst (Lead / Corresponding author)

    Research output: Contribution to journalArticle

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    Abstract

    It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and inter-bacterial competition. Anti-bacterial T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as anti-bacterial toxins and self-protecting immunity proteins able to neutralise their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell-cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of anti-bacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.
    Original languageEnglish
    Pages (from-to)2735-2749
    JournalMolecular & Cellular Proteomics
    Volume12
    Issue number10
    DOIs
    Publication statusPublished - Oct 2013

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    Bacterial Toxins
    Serratia marcescens
    Proteomics
    Phosphorylation
    Proteins
    Bacteria
    Chemical activation
    Macromolecular Substances
    Pathogens
    Substrates
    Threonine
    Phosphoric Monoester Hydrolases
    Pseudomonas aeruginosa
    Mass spectrometry
    Virulence
    Labels
    Immunity
    Mass Spectrometry
    Type VI Secretion Systems

    Cite this

    Fritsch, Maximilian J. ; Trunk, Katharina ; Alcoforado Diniz, Juliana ; Guo, Manman ; Trost, Matthias ; Coulthurst, Sarah Jane. / Proteomic identification of novel secreted anti-bacterial toxins of the Serratia marcescens Type VI secretion system. In: Molecular & Cellular Proteomics. 2013 ; Vol. 12, No. 10. pp. 2735-2749.
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    abstract = "It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and inter-bacterial competition. Anti-bacterial T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as anti-bacterial toxins and self-protecting immunity proteins able to neutralise their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell-cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of anti-bacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.",
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    Proteomic identification of novel secreted anti-bacterial toxins of the Serratia marcescens Type VI secretion system. / Fritsch, Maximilian J.; Trunk, Katharina; Alcoforado Diniz, Juliana; Guo, Manman; Trost, Matthias (Lead / Corresponding author); Coulthurst, Sarah Jane (Lead / Corresponding author).

    In: Molecular & Cellular Proteomics, Vol. 12, No. 10, 10.2013, p. 2735-2749.

    Research output: Contribution to journalArticle

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