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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

The S. marcescens T6SS Does Not Require Cell-Cell Contact for Activation and Does Not Exhibit “Dueling” Behavior(A and B) Analysis of S. marcescens Db10 expressing the TssB-mCh reporter fusion together with uniform cytoplasmic GFP. (A) Part of a representative field of view showing the red fluorescence channel (TssB-mCherry), green fluorescence channel (constitutive cytoplasmic GFP), merged red/green, and the automatically generated GFP mask. Manually identified TssB-mCh foci are highlighted with white arrows; scale bar, 1 μm. (B) Percentage of cells with 0, 1, or 2+ foci, either within the whole population (all cells) or within the subgroup of isolated cells with no touching neighbors (single cells).(C) T6SS-mediated killing of wild-type (T6SS+) or ΔtssE (T6SS mutant) S. marcescens ATCC274 target cells by different attacker strains of S. marcescens Db10, as indicated. WT, wild-type; ΔtssH, T6SS inactive mutant; ΔT6SS, mutant lacking entire T6SS; none, media only; points show mean ± SEM (n = 4).
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fig3: The S. marcescens T6SS Does Not Require Cell-Cell Contact for Activation and Does Not Exhibit “Dueling” Behavior(A and B) Analysis of S. marcescens Db10 expressing the TssB-mCh reporter fusion together with uniform cytoplasmic GFP. (A) Part of a representative field of view showing the red fluorescence channel (TssB-mCherry), green fluorescence channel (constitutive cytoplasmic GFP), merged red/green, and the automatically generated GFP mask. Manually identified TssB-mCh foci are highlighted with white arrows; scale bar, 1 μm. (B) Percentage of cells with 0, 1, or 2+ foci, either within the whole population (all cells) or within the subgroup of isolated cells with no touching neighbors (single cells).(C) T6SS-mediated killing of wild-type (T6SS+) or ΔtssE (T6SS mutant) S. marcescens ATCC274 target cells by different attacker strains of S. marcescens Db10, as indicated. WT, wild-type; ΔtssH, T6SS inactive mutant; ΔT6SS, mutant lacking entire T6SS; none, media only; points show mean ± SEM (n = 4).

Mentions: The frequency of T6SS assembly and firing, reported by formation of TssB or TssH foci, and the trigger for activation has been suggested to differ between organisms (Basler et al., 2013). In order to examine these properties for our system, we introduced the TssB-mCh reporter into a strain of S. marcescens Db10 uniformly expressing cytoplasmic GFP (TssB-mCh, ΔlacZ::GFP). Nearly 4,000 cells were imaged, with a representative partial field of view shown (Figure 3A). The number of cells in each field of view was determined from automated masks applied using the GFP signal, and TssB-mCh foci were manually identified and counted. Considering all cells, the average number of TssB foci per cell, at a given instant, was 0.35. 69% of cells had no foci, 28% had one, 3% had two, and just 0.2% (seven cells) had three (Figure 3B). Specifically considering isolated single cells with no touching neighbors, the number of foci per cell was 0.53, with 54% of cells having no foci, 39% having one focus, and 7% having two. Thus, it is clear that focus formation, and by implication T6SS activation, in S. marcescens is not dependent on cell-cell contact. In addition, a lack of any observable tendency for foci to be “paired” in neighboring cells (i.e., no “dueling”) implies at the single-cell level that there is no “Tit-for-Tat” strategy operating in S. marcescens. To further confirm that S. marcescens does not utilize this defensive regulatory strategy, we demonstrated that S. marcescens Db10 shows indistinguishable T6SS-dependent killing of T6SS-inactive versus T6SS+ S. marcescens ATCC274 target cells (Figure 3C). Hence, S. marcescens is aggressive toward even non-attacking target cells, in stark contrast with P. aeruginosa, which utilizes the defensive “Tit-for-Tat” strategy and therefore does not efficiently kill T6SS-deficient bacteria (Basler et al., 2013).


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)

The S. marcescens T6SS Does Not Require Cell-Cell Contact for Activation and Does Not Exhibit “Dueling” Behavior(A and B) Analysis of S. marcescens Db10 expressing the TssB-mCh reporter fusion together with uniform cytoplasmic GFP. (A) Part of a representative field of view showing the red fluorescence channel (TssB-mCherry), green fluorescence channel (constitutive cytoplasmic GFP), merged red/green, and the automatically generated GFP mask. Manually identified TssB-mCh foci are highlighted with white arrows; scale bar, 1 μm. (B) Percentage of cells with 0, 1, or 2+ foci, either within the whole population (all cells) or within the subgroup of isolated cells with no touching neighbors (single cells).(C) T6SS-mediated killing of wild-type (T6SS+) or ΔtssE (T6SS mutant) S. marcescens ATCC274 target cells by different attacker strains of S. marcescens Db10, as indicated. WT, wild-type; ΔtssH, T6SS inactive mutant; ΔT6SS, mutant lacking entire T6SS; none, media only; points show mean ± SEM (n = 4).
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Related In: Results  -  Collection

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fig3: The S. marcescens T6SS Does Not Require Cell-Cell Contact for Activation and Does Not Exhibit “Dueling” Behavior(A and B) Analysis of S. marcescens Db10 expressing the TssB-mCh reporter fusion together with uniform cytoplasmic GFP. (A) Part of a representative field of view showing the red fluorescence channel (TssB-mCherry), green fluorescence channel (constitutive cytoplasmic GFP), merged red/green, and the automatically generated GFP mask. Manually identified TssB-mCh foci are highlighted with white arrows; scale bar, 1 μm. (B) Percentage of cells with 0, 1, or 2+ foci, either within the whole population (all cells) or within the subgroup of isolated cells with no touching neighbors (single cells).(C) T6SS-mediated killing of wild-type (T6SS+) or ΔtssE (T6SS mutant) S. marcescens ATCC274 target cells by different attacker strains of S. marcescens Db10, as indicated. WT, wild-type; ΔtssH, T6SS inactive mutant; ΔT6SS, mutant lacking entire T6SS; none, media only; points show mean ± SEM (n = 4).
Mentions: The frequency of T6SS assembly and firing, reported by formation of TssB or TssH foci, and the trigger for activation has been suggested to differ between organisms (Basler et al., 2013). In order to examine these properties for our system, we introduced the TssB-mCh reporter into a strain of S. marcescens Db10 uniformly expressing cytoplasmic GFP (TssB-mCh, ΔlacZ::GFP). Nearly 4,000 cells were imaged, with a representative partial field of view shown (Figure 3A). The number of cells in each field of view was determined from automated masks applied using the GFP signal, and TssB-mCh foci were manually identified and counted. Considering all cells, the average number of TssB foci per cell, at a given instant, was 0.35. 69% of cells had no foci, 28% had one, 3% had two, and just 0.2% (seven cells) had three (Figure 3B). Specifically considering isolated single cells with no touching neighbors, the number of foci per cell was 0.53, with 54% of cells having no foci, 39% having one focus, and 7% having two. Thus, it is clear that focus formation, and by implication T6SS activation, in S. marcescens is not dependent on cell-cell contact. In addition, a lack of any observable tendency for foci to be “paired” in neighboring cells (i.e., no “dueling”) implies at the single-cell level that there is no “Tit-for-Tat” strategy operating in S. marcescens. To further confirm that S. marcescens does not utilize this defensive regulatory strategy, we demonstrated that S. marcescens Db10 shows indistinguishable T6SS-dependent killing of T6SS-inactive versus T6SS+ S. marcescens ATCC274 target cells (Figure 3C). Hence, S. marcescens is aggressive toward even non-attacking target cells, in stark contrast with P. aeruginosa, which utilizes the defensive “Tit-for-Tat” strategy and therefore does not efficiently kill T6SS-deficient bacteria (Basler et al., 2013).

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