Stretching the arms of the type VI secretion sheath protein

Bacteria use the T6SS to modify the environment to their advantage. The T6SS can translocate many different effectors into the surrounding medium, as well as into the periplasm and cytoplasm of neighboring cells. Effector proteins can be attached to a spike‐shaped complex positioned at the tip of the tube and/or are loaded into the internal tube. The diversity and efficiency of T6SS effectors render the system to be extremely versatile and are essential for the survival of organisms carrying T6SS organelles.

One of the specific peculiarities of the T6SS is the selection mechanism of secreted effectors. T6SS effectors do not carry a unique motif in their amino acid sequences, suggesting that the selection takes place at the level of three‐dimensional structure, which is more conserved than the sequence. This is exemplified by the PAAR‐repeat proteins that contain three Proline‐Alanine‐Alanine‐aRginine (PAAR) motifs that do not encode a binding site, but instead give the polypeptide chain a threefold symmetric structure 4. The actual interaction site of PAAR‐repeat proteins with the T6SS organelle—the tip of the VgrG spike protein in this case—carries no sequence similarity. Presently, PAAR‐repeat motifs remain the best and strongest indication that a protein is secreted by the T6SS. Thus, the knowledge of the three‐dimensional structure of the T6SS organelle is paramount to understanding not only how the system works but also how it selects the substrates for secretion.

To this end, studies of T6SS structure showed that its tube subunit 5, its VgrG/PAAR spike 4, and the sheath 6, 7 are homologous to their phage and R‐type pyocin counterparts 8. These findings put forward a mechanism of sheath contraction‐driven secretion. However, the role of sheath contraction in effector secretion has never been rigorously shown. Furthermore, the structure of the T6SS sheath in the extended state and that of the T6SS baseplate, both of which are essential for the contraction‐driven secretion mechanism, remained unknown.

To address these questions, Brackmann et al first examined the structure of the R‐type pyocin 8. The pyocin sheath subunit comprises the conserved core of the T6SS subunit 8, which, in addition to being larger, consists of two polypeptide chains—a larger VipA and smaller VipB component. The subunits of both sheaths are connected to each other in the same manner via long N‐ and C‐terminal linkers that form a two‐dimensional mesh akin to a protective bottle sleeve. In the contracted state, the N‐terminal linkers are fully stretched and the sheath subunits are densely packed by complementary electrostatic charges 1.

To examine the role of charge complementarity for contraction, Brackmann et al changed the electrostatics of the interacting surfaces of the VipB sheath subunit by introducing K223A and D333A mutations 1. The dynamics of sheath contraction in these mutants were visualized in vivo by labeling the VipA subunit with GFP. The newly assembled T6SS structures appeared as long fluorescent rods spanning the cell. These rods could spontaneously contract and were quickly disassembled by a special ATPase called ClpV. T6SS killing capacity was assessed by measuring the survival of Escherichia coli prey cells incubated with Vibrio cholerae. VipB‐K223A mutant sheaths were defective in assembly, while VipB‐D333A‐assembled wild‐type‐like structures did not contract in vivo but did so during the purification procedure. The D333 mutant showed no T6SS‐mediated prey killing. These experiments confirm that the charge complementarity of the contracted state interfaces is important for sheath contraction.

Brackmann et al further hypothesized that extending the length of the N‐terminal linker of the VipA T6SS sheath subunit should disrupt the contraction because these linkers are fully stretched in the contracted state. Indeed, adding as few as two extra amino acids to the linker blocks the contraction and results in a non‐functional phenotype (Fig 1).

The most amazing outcome of these manipulations is that non‐contractile structures were stable enough to be purified and analyzed by mass spectrometry and cryoEM, which showed that these complexes carry the T6SS baseplate, and the sheath capping protein TssA 1, 3. This made it possible to obtain the structure of the T6SS in the extended, pre‐secretion state 2, 3. Although the resolution was insufficient for exact interpretation in terms of protein components, it clearly showed that the T6SS baseplate follows the phage baseplate architecture (e.g., of the phage T4 9) with a centrally positioned VgrG–PAAR spike complex surrounded by six wedges that contain a trimeric protein (TssK) at their periphery 3. The capping complex on the opposite end of the T6SS structure was poorly resolved, leaving the question of the location and function of the elusive TssA protein open 3.

Helical symmetry of the sheath tube part of the structure was utilized to obtain a 3.7 Å resolution cryoEM map and to build atomic models of the extended sheath and the complete tube for a mutant carrying three extra residues in the N‐terminal linker of the VipA sheath subunit 2. The extension of the linker arm turned out to alter the assembly of the sheath. The N‐terminal linker no longer extended to a subunit in the neighboring baseplate‐proximal layer (or ring) of the sheath, but instead connected to a subunit in the same layer. A sheath with such a topology could not switch to a contracted state, because the N‐terminal linker of VipA had to be even longer.

Extension of the N‐terminal linker by one amino acid resulted in a wild‐type‐like phenotype in terms of sheath contraction dynamics and T6SS‐mediated prey killing. Extension by two amino acids showed no sheath contraction in vivo and no T6SS‐mediated prey killing. Such sheaths contracted upon purification (just like the wild type) and their structure and subunit connectivity is identical to wild type 2. Thus, the original hypothesis that extension of linkers connecting sheath subunits into a two‐dimensional mesh would stop the sheath from contracting turns out to be correct, although for extensions greater than three amino acids the contraction is blocked by an altered assembly instead of the linker length per se.

Non‐contractile sheaths contained the inner tube formed by the Hcp protein 1, which made it possible to determine the structure of the T6SS tube in its fully assembled form 2. The tube was found to have the same symmetry as the extended sheath, further confirming T6SS homology to phage tail/pyocin systems. The interactions between the sheath and tube subunits are similar and utilize equivalent structural elements as in the pyocin 8. In phages, the sheath is assembled onto the tube starting from the baseplate and utilizing the latter as a scaffold. The same mechanism was suggested to be employed by the T6SS organelle. This hypothesis has recently been confirmed in a series of clever imaging experiments 10.

In summary, the papers by Brackmann et al 1, Wang et al 2, and Nazarov et al 3 show that a such a complex, multicomponent, and dynamic structure as the T6SS requires many different experimental approaches to confirm or disprove a hypothesis that specifically addresses its functional mechanism. A lack‐of‐function phenotype can be masked by incorrect assembly, and the studied biological object may appear wild type‐like in analyses that do not take into account perturbations to its atomic structure.