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Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

Gerc AJ, Diepold A, Trunk K, Porter M, Rickman C, Armitage JP, Stanley-Wall NR, Coulthurst SJ - Cell Rep (2015)

Bottom Line: The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells.Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo.We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

No MeSH data available.


Related in: MedlinePlus

Visualization of Distinct Components within an Active Type VI Secretion System in Serratia marcescens(A) Cartoon depiction of the T6SS with the components visualized in this study highlighted in red. The fourteen core components and one accessory component, Fha, are labeled. Cytoplasm (cyto), periplasm (peri), inner membrane (IM), and outer membrane (OM) of the secreting cell are indicated.(B) T6SS-dependent secretion of Hcp and the effector Ssp1 by S. marcescens Db10 (WT) and derivatives expressing fusions of mCherry to the C terminus of TssB (TssB-mCh), TssH (TssH-mCh), TssJ (TssJ-mCh), or TssL (TssL-mCh). The T6SS inactive mutant ΔtssE is a negative control, and cellular (cell) and secreted (sec) fractions were subjected to immunoblotting using anti-Hcp and anti-Ssp1 antisera as indicated.(C) T6SS-dependent antibacterial activity of fluorescent reporter strains against P. fluorescens target cells. Recovery of target cells following a 4-hr co-culture with the attacking strains of S. marcescens indicated; points show mean ± SEM (n = 4).(D–G) Representative images of cells expressing TssB-mCh (D), TssH-mCh (E), TssJ-mCh (F), or TssL-mCh (G). Upper panels: DIC images; lower panels: corresponding fluorescence images (mCherry channel); scale bar, 1 μm.See also Figure S1.
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fig1: Visualization of Distinct Components within an Active Type VI Secretion System in Serratia marcescens(A) Cartoon depiction of the T6SS with the components visualized in this study highlighted in red. The fourteen core components and one accessory component, Fha, are labeled. Cytoplasm (cyto), periplasm (peri), inner membrane (IM), and outer membrane (OM) of the secreting cell are indicated.(B) T6SS-dependent secretion of Hcp and the effector Ssp1 by S. marcescens Db10 (WT) and derivatives expressing fusions of mCherry to the C terminus of TssB (TssB-mCh), TssH (TssH-mCh), TssJ (TssJ-mCh), or TssL (TssL-mCh). The T6SS inactive mutant ΔtssE is a negative control, and cellular (cell) and secreted (sec) fractions were subjected to immunoblotting using anti-Hcp and anti-Ssp1 antisera as indicated.(C) T6SS-dependent antibacterial activity of fluorescent reporter strains against P. fluorescens target cells. Recovery of target cells following a 4-hr co-culture with the attacking strains of S. marcescens indicated; points show mean ± SEM (n = 4).(D–G) Representative images of cells expressing TssB-mCh (D), TssH-mCh (E), TssJ-mCh (F), or TssL-mCh (G). Upper panels: DIC images; lower panels: corresponding fluorescence images (mCherry channel); scale bar, 1 μm.See also Figure S1.

Mentions: The T6SS is a large macromolecular assembly spanning the bacterial cell envelope and whose mode of action is related to the injection mechanism of contractile bacteriophage tails. Recent work has revealed key aspects of the organization and mechanism of the T6SS, but the picture is far from complete. According to current models (Ho et al., 2014; Zoued et al., 2014), the T6SS is built using fourteen “core” components that form several subassemblies. An extracellular puncturing device, which is fired from the cell, is made up of a tube of Hcp (TssD) with a trimer of VgrG (TssI) at its distal end, further sharpened by a PAAR protein at the tip (Brunet et al., 2014; Shneider et al., 2013). A membrane complex, made up of the integral inner membrane proteins TssL and TssM and the outer membrane lipoprotein TssJ, anchors a cytoplasmic baseplate-like structure at the cell envelope. Upon this basal complex, a contractile tubular sheath made of TssBC subunits assembles in the cytoplasm, around the Hcp-VgrG structure (Basler et al., 2012; Brunet et al., 2014; Zoued et al., 2014) (Figure 1A). Prior to firing, this TssBC sheath is in an extended conformation. Contraction of the TssBC sheath then propels the puncturing device through the basal complex, out of the cell, and into an adjacent target cell. The contracted TssBC sheath is recognized by the AAA+ ATPase, TssH (ClpV), which disassembles the sheath, allowing recycling of the TssBC subunits and the components of the basal complex (Basler and Mekalanos, 2012; Kapitein et al., 2013; Kube et al., 2014). Effectors are translocated by covalent or non-covalent association with different components of the puncturing device (Dong et al., 2013; Shneider et al., 2013; Silverman et al., 2013; Whitney et al., 2014).


Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

Gerc AJ, Diepold A, Trunk K, Porter M, Rickman C, Armitage JP, Stanley-Wall NR, Coulthurst SJ - Cell Rep (2015)

Visualization of Distinct Components within an Active Type VI Secretion System in Serratia marcescens(A) Cartoon depiction of the T6SS with the components visualized in this study highlighted in red. The fourteen core components and one accessory component, Fha, are labeled. Cytoplasm (cyto), periplasm (peri), inner membrane (IM), and outer membrane (OM) of the secreting cell are indicated.(B) T6SS-dependent secretion of Hcp and the effector Ssp1 by S. marcescens Db10 (WT) and derivatives expressing fusions of mCherry to the C terminus of TssB (TssB-mCh), TssH (TssH-mCh), TssJ (TssJ-mCh), or TssL (TssL-mCh). The T6SS inactive mutant ΔtssE is a negative control, and cellular (cell) and secreted (sec) fractions were subjected to immunoblotting using anti-Hcp and anti-Ssp1 antisera as indicated.(C) T6SS-dependent antibacterial activity of fluorescent reporter strains against P. fluorescens target cells. Recovery of target cells following a 4-hr co-culture with the attacking strains of S. marcescens indicated; points show mean ± SEM (n = 4).(D–G) Representative images of cells expressing TssB-mCh (D), TssH-mCh (E), TssJ-mCh (F), or TssL-mCh (G). Upper panels: DIC images; lower panels: corresponding fluorescence images (mCherry channel); scale bar, 1 μm.See also Figure S1.
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Related In: Results  -  Collection

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fig1: Visualization of Distinct Components within an Active Type VI Secretion System in Serratia marcescens(A) Cartoon depiction of the T6SS with the components visualized in this study highlighted in red. The fourteen core components and one accessory component, Fha, are labeled. Cytoplasm (cyto), periplasm (peri), inner membrane (IM), and outer membrane (OM) of the secreting cell are indicated.(B) T6SS-dependent secretion of Hcp and the effector Ssp1 by S. marcescens Db10 (WT) and derivatives expressing fusions of mCherry to the C terminus of TssB (TssB-mCh), TssH (TssH-mCh), TssJ (TssJ-mCh), or TssL (TssL-mCh). The T6SS inactive mutant ΔtssE is a negative control, and cellular (cell) and secreted (sec) fractions were subjected to immunoblotting using anti-Hcp and anti-Ssp1 antisera as indicated.(C) T6SS-dependent antibacterial activity of fluorescent reporter strains against P. fluorescens target cells. Recovery of target cells following a 4-hr co-culture with the attacking strains of S. marcescens indicated; points show mean ± SEM (n = 4).(D–G) Representative images of cells expressing TssB-mCh (D), TssH-mCh (E), TssJ-mCh (F), or TssL-mCh (G). Upper panels: DIC images; lower panels: corresponding fluorescence images (mCherry channel); scale bar, 1 μm.See also Figure S1.
Mentions: The T6SS is a large macromolecular assembly spanning the bacterial cell envelope and whose mode of action is related to the injection mechanism of contractile bacteriophage tails. Recent work has revealed key aspects of the organization and mechanism of the T6SS, but the picture is far from complete. According to current models (Ho et al., 2014; Zoued et al., 2014), the T6SS is built using fourteen “core” components that form several subassemblies. An extracellular puncturing device, which is fired from the cell, is made up of a tube of Hcp (TssD) with a trimer of VgrG (TssI) at its distal end, further sharpened by a PAAR protein at the tip (Brunet et al., 2014; Shneider et al., 2013). A membrane complex, made up of the integral inner membrane proteins TssL and TssM and the outer membrane lipoprotein TssJ, anchors a cytoplasmic baseplate-like structure at the cell envelope. Upon this basal complex, a contractile tubular sheath made of TssBC subunits assembles in the cytoplasm, around the Hcp-VgrG structure (Basler et al., 2012; Brunet et al., 2014; Zoued et al., 2014) (Figure 1A). Prior to firing, this TssBC sheath is in an extended conformation. Contraction of the TssBC sheath then propels the puncturing device through the basal complex, out of the cell, and into an adjacent target cell. The contracted TssBC sheath is recognized by the AAA+ ATPase, TssH (ClpV), which disassembles the sheath, allowing recycling of the TssBC subunits and the components of the basal complex (Basler and Mekalanos, 2012; Kapitein et al., 2013; Kube et al., 2014). Effectors are translocated by covalent or non-covalent association with different components of the puncturing device (Dong et al., 2013; Shneider et al., 2013; Silverman et al., 2013; Whitney et al., 2014).

Bottom Line: The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells.Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo.We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

No MeSH data available.


Related in: MedlinePlus