T6SS and contractile phage share a number of core structural components. Homologous components are depicted in the same color. While phage attach to the outer membrane (OM) via tail fibers connected to its baseplate, T6SS baseplate attaches to an inner membrane (IM) complex that spans the periplasm and associates with the outer membrane. Contraction of phage sheath delivers the phage spike into a target cell, while contraction of T6SS sheath forces the T6SS spike out of the cell and potentially across a target membrane (TM). Proteins conserved in T4 phage and the T6SS are labeled.

(A) Baseplate complex forms consisting of TssE, TssJ, TssK, TssL and TssM. Other components not pictured include TssA, TssF, and TssG. In some T6SSs, Fha is an essential part of this complex. TssJ, TssK, TssL and TssM are placed in this drawing based on protein localization and interaction studies (; ), while TssE position is inferred from phage homolog () (B) VgrG, PAAR, and effector proteins are recruited to this complex and assemble into the structure. VgrG interaction with PAAR or effectors likely contributes to the overall stability of the apparatus assembly. Although these components are pictured here as being cytosolic, it is unclear whether there is an opening into the periplasm. (C) Hcp tube polymerizes from VgrG while the VipA/VipB sheath polymerizes around it. (D) Analogous to phage, a conformation change in the sheath structure results in a contraction event that launches the Hcp tube out of the cell and across a target membrane. This contraction event delivers the loaded VgrG-effector “warhead” through the layers of the cell envelope, however it is not known how often penetration into the cytosol occurs, if at all. It is also unknown how much Hcp is lost outside the cell and how much is retained within the cytosol. (E) ClpV uses ATP to remodel the contracted sheath, restoring the pool of available sheath subunits. The now unsheathed Hcp tube disassembles; parts of the tube that not expelled from the cell are recycled within the cytosol. (F) The naked baseplate complex is then ready to be recycled or disassembled, depending on the T6SS and its activation state.

Spatial geometry of anti-bacterial T6SS attacks. (A) Fully assembled T6SS tube (orange arrow) and sheath (purple rectangle) can extends across the diameter of the cell. By comparison, T4 phage tails are typically only ~100 nm (~1/10 cell width). Phage tail length is drawn approximately to scale. (B) After contraction, sheath length is reduced by close to 50%. Assuming the inner Hcp completely fills the sheath, the full range of the T6SS (orange halo) is a zone approximately 500 nm wide (~1/2 cell width) surrounding the cell. (C) Assuming perfectly cylindrical cellular geometry and tight spatial packing, at most 1/6th of the T6SS⁺ attack range will contain a given prey cell. This number is even smaller the larger the distance between the T6SS⁺ predator and the prey cell, highlighting the need for proper aiming of T6SS attacks. If a prey cell cannot be sensed, the attacker must fire repeatedly in all directions, wasting a majority of the attack potential.

T6SS effectors are loaded onto the VgrG spike complex or within the distal end of the Hcp tube (left panel). Sheath contraction leads to the simultaneous delivery of the VgrG spike and all associated effectors (middle panel), abrogating the need for the Hcp tube to be stably maintained (right panel). Prototypical examples of effector classes 1–3 have been characterized: V. cholerae VgrG-1 and VgrG-3 (Class 1), V. cholerae TseL (Class 2), D. dadantii RhsA (Class 3). Effector classes 4 and 5 represent hypothetical mechanisms that are likely to exist. Class 4 effectors associate with PAAR protein extensions such as transthyretin-like domains () or Rhs-repeat domains (). Class 5 effectors bind to the lumenal side of the Hcp tube; although it has not yet been experimentally confirmed, it is likely that P. aeruginosa effector Tse2 falls into this category.

T6SS counterattack sensing pathway. (A) The program Hmmsearch () was used to identify 761 VipA (pfam05591)/VipB (pfam05943)-containing gene loci identified in sequenced bacterial genomes (ftp://ftp.ncbi.nih.gov/genomes/bacteria). 128 of these loci contained nearby (within 40kb) Fha (pfam00498), PpkA (PF00069 or PF13519), and PppA (PF13672) genes. Subsets of these loci carrying different combinations of TagQ (pfam13488), TagR (pfam03781), TagS (pfam02687), or TagT (pfam00005) homologs were also identified. All loci with TagS also had TagT (*). (B and C) Various signals involving membrane perturbation including exogenous T6SS attack, T4SS mating pair formation and certain membrane disrupting antibiotics like polymyxin B can trigger Fha phosphorylation by PpkA. TagR directly activates PpkA, while TagQ positions TagR in the periplasm and associates it with the outer membrane. TagS and TagT comprise an inner membrane ABC transporter. It is possible that TagS and TagT directly sense the membrane perturbation signal (B) or are responsible for localization of the actual signal sensor (C). It should be noted that TagS and TagT are not required for delivery of TagQ and TagR to the periplasmic space ().