PMID:22367545

Citation	Basler, M, Pilhofer, M, Henderson, GP, Jensen, GJ and Mekalanos, JJ (2012) Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483:182-6
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.
Links	PubMed PMC3527127 Online version:10.1038/nature10846
Keywords	Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Bacterial Proteins/ultrastructure; Bacterial Secretion Systems/physiology; Bacteriophages/chemistry; Bacteriophages/physiology; Cell Membrane/metabolism; Cryoelectron Microscopy; Electron Microscope Tomography; Microscopy, Fluorescence; Vibrio cholerae/chemistry; Vibrio cholerae/cytology; Vibrio cholerae/metabolism; Vibrio cholerae/ultrastructure
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Significance

The T6SS of V. cholerae possess antibacterial activity against E. coli that correlates with its ability to secrete protein Hcp. In this study researchers showed that protein secretion by the type VI secretion system of Vibrio cholerae correlates with the activity of a dynamic intracellular structure that leads to T6SS-dependent secretion of Hcp and killing of E-coli. This structure appears structurally and functionally homologous to contractile phage tail sheath.

Figure 1: Fluorescence light microscopy of VipA-sfGFP

• To test the hypothesis that the T6SS is a dynamic structure, C-terminal fusion of VipA protein with super-folder green fluorescent protein (sfGFP) was constructed and expressed in pBAD24 plasmid.
• The cells expressing the functional sfGFP fusion protein revealed the following:

1. VipA–sfGFP fusion is associated with long straight structures in the cytosol.
2. The number of visible structures in a single cell varied from 0 to 5 in wild-type.
3. These structures were not visible in vipB mutant cells.
4. The sheath structures were highly dynamic.
5. the sheaths underwent extension-contraction-disassembly cycles in both wild type and vipA mutant cells.
6. The sheaths assembled at speeds of 20–30 ms. Contraction was very fast, occurring in about 5 ms or less. Then the sheaths disassembled over the next 30–60 s.

Figure 2: Electron cryotomographic imaging of T6SS structure inside intact cells

• Electron cryomography (ECT) was used to visualize the bacterial cytoskeletal structure directly in 3D.
• ECT revealed 2 distinct conformations for the sheaths: extended and contracted.
• Both conformations were located in the cytosol and roughly perpendicular to the cytoplasmic membrane.
• Tubular structures were observed in wild type cells.
• Tubular structures were missing in the vipB mutant, VCA109 mutant, and VCA0109/ClpV double mutant.
• Extended conformations had lengths of 667+/-83 nm, and diameters of 11.6+/-0.7nm.
• Contracted conformations had lengths of 372+/-56nm, and diameters of 14.6+/-0.7nm.
• The tubular structures of both conformations were roughly perpendicular to the cytoplasmic membrane
• The tubes connected to the membrane by a bell-shaped base.

Figure 3: Images of purified VipA/VipB sheaths and comparison with phage tails

• To test the hypothesis that the dynamic structured observed in VipA-sfGFP expressing cells (Fig. 1) and the tubes observed by ECT (Fig. 2) were indeed T6SS sheath structures, the VipA/VipB sheaths were purified and the negative stain electron microscopic images were analyzed:

1. Straight, hollow tubular structures were observed.
2. No tubular structures were observed in VipA or VipB mutants.
3. Similar to the in vivo experiments, both contracted and extended conformations were recognized.
4. Purified sheaths showed helical surface ridges, as well as cogwheel-like cross sections with 12 paddles per rotation. This structure is identical to contracted T4 phage sheaths.

• Mass spec analysis of the proteins in the purified samples revealed the presence of VipA and VipB (the most abondent proteins), as well as ClpV (which interacts directly with VipB, most strongly in VipA/VipB heterodimer structures), and VCA0109 (a homologue of T4 base-plate protein gp25).

• Hcp was not found in purified contracted T6SS sheaths. Therefore, it was concluded that Hcp is expelled from the cell at contraction.

Figure 4: Model of T6SS action

• Based on all the above observations, the authors proposed the following model of action for T6SS:

• This model is based on ClpV and T6SS sheath recycling and consists of the 4 following phases:

1. Assembly: Which occurs in 2 steps:

• Step1: Formation of the base-plate complex which is composed of a bell-shaped cytoplasmic component (gp25, VgrG and other T6SS proteins), and a periplasmic component. This would initiate the polymerization of Hcp Tube.
• Step 2: Polymerization of the sheath from VipA and VipB heterodimers around the Hcp tube.

1. Extention: This is the "ready to fire" conformation where the Hcp VipA/VipB tube formation is completed.
2. Contraction: Which takes place in the following order:
   1. An extracellular signal.
   2. A conformational change in the base plate complex.
   3. Sheath contraction.
   4. Translocation (secretion) of the VgrG/Hcp tube complex through effector cell membranes and penetration of the target cell membrane.
3. Disassembly: Disassembly of the contracted sheath by ClpV ATPase and recycling the VipA/VipB dimers.

Annotations

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References

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