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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

Simultaneous Observation of Two Type VI Components Reveals Differing Associations of TssH, TssJ, and TssL with TssB(A) Hcp secretion by S. marcescens Db10 (WT) and derivatives expressing a fusion of GFP to the C terminus of TssB (TssB-GFP), or expressing both TssB-GFP and a fusion of mCherry with TssH (TssH-mCh, TssB-GFP), TssJ (TssJ-mCh, TssB-GFP), or TssL (TssL-mCh, TssB-GFP). Cellular (cell) and secreted (sec) fractions subjected to anti-Hcp immunoblot, with the ΔtssE mutant as a negative control.(B) Representative fluorescence images from cells expressing TssH-mCh and TssB-GFP (top), TssJ-mCh and TssB-GFP (middle), or TssL-mCh and TssB-GFP (bottom). Indicated parts of the micrographs are magnified in the insets. Panels from left to right: GFP (TssB-GFP) channel, mCherry (TssX-mCh) channel, and a false-colored merge. Scale bars, 1 μm.
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fig5: Simultaneous Observation of Two Type VI Components Reveals Differing Associations of TssH, TssJ, and TssL with TssB(A) Hcp secretion by S. marcescens Db10 (WT) and derivatives expressing a fusion of GFP to the C terminus of TssB (TssB-GFP), or expressing both TssB-GFP and a fusion of mCherry with TssH (TssH-mCh, TssB-GFP), TssJ (TssJ-mCh, TssB-GFP), or TssL (TssL-mCh, TssB-GFP). Cellular (cell) and secreted (sec) fractions subjected to anti-Hcp immunoblot, with the ΔtssE mutant as a negative control.(B) Representative fluorescence images from cells expressing TssH-mCh and TssB-GFP (top), TssJ-mCh and TssB-GFP (middle), or TssL-mCh and TssB-GFP (bottom). Indicated parts of the micrographs are magnified in the insets. Panels from left to right: GFP (TssB-GFP) channel, mCherry (TssX-mCh) channel, and a false-colored merge. Scale bars, 1 μm.

Mentions: To directly compare the localization of TssH, TssJ, and TssL with the reference TssB, functional dual reporter strains were constructed. The chromosomally encoded TssH-mCh, TssJ-mCh, and TssL-mCh fusions were each combined with a tssB-GFP allele for co-expression of a TssB-GFP fusion protein from the normal chromosomal location. As for the single TssB-,TssH-,TssJ-, and TssL-mCh fusion strains, strains of S. marcescens Db10 expressing TssB-GFP alone or any of the TssH-,TssJ-,TssL-mCh, or TssB-GFP dual reporters retained the ability to secrete Hcp, confirming their T6SS functionality (Figure 5A). Imaging the three dual reporter strains revealed distinct patterns of association with TssB foci (Figure 5B). TssH foci were found to be strongly co-localized with TssB foci, whereas TssB foci were often found without corresponding TssH foci (top row). TssJ is localized much less specifically around the cell, and specific co-localization of TssJ with TssB was not observed (middle row), although TssJ sometimes appeared enriched around TssB foci. The association between TssL and TssB followed a different pattern again. TssB foci were highly co-localized with TssL foci, but many additional TssL foci without TssB were also observed (bottom row). Quantitative co-localization by a Pearson’s correlation method (comparing each channel over every pixel) gave similar correlation values (between 0.48 and 0.58) for each of TssH, TssJ, and TssL with TssB, presumably because all show some diffuse non-focal fluorescence in addition to any foci. However, given that the foci are of primary interest, a more suitable, object-based approach was adopted.


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)

Simultaneous Observation of Two Type VI Components Reveals Differing Associations of TssH, TssJ, and TssL with TssB(A) Hcp secretion by S. marcescens Db10 (WT) and derivatives expressing a fusion of GFP to the C terminus of TssB (TssB-GFP), or expressing both TssB-GFP and a fusion of mCherry with TssH (TssH-mCh, TssB-GFP), TssJ (TssJ-mCh, TssB-GFP), or TssL (TssL-mCh, TssB-GFP). Cellular (cell) and secreted (sec) fractions subjected to anti-Hcp immunoblot, with the ΔtssE mutant as a negative control.(B) Representative fluorescence images from cells expressing TssH-mCh and TssB-GFP (top), TssJ-mCh and TssB-GFP (middle), or TssL-mCh and TssB-GFP (bottom). Indicated parts of the micrographs are magnified in the insets. Panels from left to right: GFP (TssB-GFP) channel, mCherry (TssX-mCh) channel, and a false-colored merge. Scale bars, 1 μm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594159&req=5

fig5: Simultaneous Observation of Two Type VI Components Reveals Differing Associations of TssH, TssJ, and TssL with TssB(A) Hcp secretion by S. marcescens Db10 (WT) and derivatives expressing a fusion of GFP to the C terminus of TssB (TssB-GFP), or expressing both TssB-GFP and a fusion of mCherry with TssH (TssH-mCh, TssB-GFP), TssJ (TssJ-mCh, TssB-GFP), or TssL (TssL-mCh, TssB-GFP). Cellular (cell) and secreted (sec) fractions subjected to anti-Hcp immunoblot, with the ΔtssE mutant as a negative control.(B) Representative fluorescence images from cells expressing TssH-mCh and TssB-GFP (top), TssJ-mCh and TssB-GFP (middle), or TssL-mCh and TssB-GFP (bottom). Indicated parts of the micrographs are magnified in the insets. Panels from left to right: GFP (TssB-GFP) channel, mCherry (TssX-mCh) channel, and a false-colored merge. Scale bars, 1 μm.
Mentions: To directly compare the localization of TssH, TssJ, and TssL with the reference TssB, functional dual reporter strains were constructed. The chromosomally encoded TssH-mCh, TssJ-mCh, and TssL-mCh fusions were each combined with a tssB-GFP allele for co-expression of a TssB-GFP fusion protein from the normal chromosomal location. As for the single TssB-,TssH-,TssJ-, and TssL-mCh fusion strains, strains of S. marcescens Db10 expressing TssB-GFP alone or any of the TssH-,TssJ-,TssL-mCh, or TssB-GFP dual reporters retained the ability to secrete Hcp, confirming their T6SS functionality (Figure 5A). Imaging the three dual reporter strains revealed distinct patterns of association with TssB foci (Figure 5B). TssH foci were found to be strongly co-localized with TssB foci, whereas TssB foci were often found without corresponding TssH foci (top row). TssJ is localized much less specifically around the cell, and specific co-localization of TssJ with TssB was not observed (middle row), although TssJ sometimes appeared enriched around TssB foci. The association between TssL and TssB followed a different pattern again. TssB foci were highly co-localized with TssL foci, but many additional TssL foci without TssB were also observed (bottom row). Quantitative co-localization by a Pearson’s correlation method (comparing each channel over every pixel) gave similar correlation values (between 0.48 and 0.58) for each of TssH, TssJ, and TssL with TssB, presumably because all show some diffuse non-focal fluorescence in addition to any foci. However, given that the foci are of primary interest, a more suitable, object-based approach was adopted.

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