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Type VI secretion requires a dynamic contractile phage tail-like structure

Abstract

Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

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Figure 1: Fluorescence light microscopy of VipA–sfGFP.
Figure 2: Electron cryotomographic imaging of T6SS structures inside intact cells.
Figure 3: Images of purified VipA/VipB sheaths and comparison with phage tails.
Figure 4: Model of T6SS action.

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Acknowledgements

We thank T. G. Bernhardt and N. T. Peters for assistance with fluorescence microscopy, discussions and for a gift of plasmids carrying sfGFP and mCherry2 genes. We thank the Nikon Imaging Center at Harvard Medical School for help with fluorescence microscopy, and Research Precision Instruments and Hamamatsu for lending an ORCA-Flash2.8 camera. We thank the Harvard Medical School Electron Microscopy Facility for help with and supervision of transmission electron microscopy. We thank M. K. Waldor for a V. cholerae 2740-80 strain and discussions. We thank D. Ewen Cameron for a knockout construct pWM91-flgG. We thank B. Wen and Z. Li for initial cryotomographic studies. This work was supported by National Institute of Allergy and Infectious Diseases grants AI-018045 and AI-26289 to J.J.M. and National Institute of General Medical Sciences grant GM094800B to G.J.J.

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Authors

Contributions

All authors helped design and analyse experiments; M.B., M.P. and G.P.H. performed experiments, and M.B., M.P., G.J.J. and J.J.M. wrote the paper.

Corresponding authors

Correspondence to G. J. Jensen or J. J. Mekalanos.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 with legends, Supplementary Tables 1-2 and full legends for Supplementary Movies 1-11. (PDF 13211 kb)

Supplementary Movie 1

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP dynamics, high magnification. (MOV 9861 kb)

Supplementary Movie 2

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP dynamics. (MOV 6278 kb)

Supplementary Movie 3

This movie shows a high speed time-lapse fluorescence microscopy of VipA-sfGFP contraction. (MOV 1797 kb)

Supplementary Movie 4

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP dynamics at different expression levels of VipA-sfGFP in ∆VipA background. (MOV 6114 kb)

Supplementary Movie 5

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP dynamics at different expression levels of VipA-sfGFP in wild-type background. (MOV 7600 kb)

Supplementary Movie 6

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP dynamics in V52 strain. (MOV 2640 kb)

Supplementary Movie 7

This movie shows a time-lapse fluorescence microscopy of VipA-mCherry2 dynamics. (MOV 2052 kb)

Supplementary Movie 8

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP in ∆ClpV background. (MOV 5195 kb)

Supplementary Movie 9

This movie shows a time-lapse fluorescence microscopy of VipA-sfGFP in ∆VCA0109 background. (MOV 5316 kb)

Supplementary Movie 10

In this movie we see a 3-D tomographic analysis showing that T6SS tubular structures are clearly located in the cytoplasm. (MOV 28615 kb)

Supplementary Movie 11

This movie file contains a narrated animation summarizing the light and electron microscopy data and depicting our model for the assembly, firing, and disassembly of the T6SS. (MOV 26970 kb)

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Basler, M., Pilhofer, M., Henderson, G. et al. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186 (2012). https://doi.org/10.1038/nature10846

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