Diverse roles of TssA‐like proteins in the assembly of bacterial type VI secretion systems

Abstract

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA‐like or TagA‐like proteins with a conserved N‐terminal domain and varying C‐terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N‐terminal domain, their roles in T6SS biogenesis are fundamentally different.

Novel structural components generate distinct type VI secretion system anchoring modes

“…1EF) consistent with the fact that P. putida tssA1 has been reported to be essential for K1-T6SS function (31) and that in other organisms sheath formation is only observed in vivo when TssA is present (27). As it has been demonstrated that long TssA proteins from E. coli , V. cholerae and P. aeruginosa (H2-T6SS) initially localize at the baseplate and subsequently migrate at the tip of the growing sheath (27, 29), we wanted to investigate if a short TssA protein, like P. putida TssA1, exhibits a similar behavior. To monitor its localization during sheath assembly, we generated a P. putida rpoN strain expressing sfGFP-TssA1 and TssB1-mScarlet-I from their native loci.…”

“…Average protein sizes in kDa are given for T6SS components of each phylogenetic group and the domain architecture of TssA-like proteins is shown; the N-terminus of all TssA-like proteins forms an ImpA domain whilst their C-terminus varies (28, 29). “TagA” refers to homologues of the E. coli TagA anchor protein (30), “TagA V ” has been described as the anchor for the T6SS of V. cholerae (29), and “TagA O ”, which has a different C-terminus compared to the other two forms of TagA, has not been described before. Long TssA proteins (TssA L ) are encoded in T6SS clusters from phylogenetic groups 1 and 2, whilst short TssA proteins (TssA S ) are encoded in T6SS clusters from phylogenetic groups 3-5.…”

“…Representative examples of well-studied T6SSs from each phylogenetic group are: P. aeruginosa H2-T6SS and V. cholerae T6SS (group 1); EAEC T6SS (group 2); P. aeruginosa H1-T6SS (group 3); P. aeruginosa H3-T6SS (group 4A); P. putida K1-T6SS (group 4B); Agrobacterium tumefaciens T6SS (group 5). TssA L proteins usually co-occur with TagA-like components (only one TagA protein and its cognate TssA partner are encoded in each cluster), but examples of T6SS clusters belonging to phylogenetic groups 1 and 2 encoding a TssA L without containing a tagA gene also exist (for example the H2-T6SS of P. aeruginosa in phylogenetic group 1 (29)). TssA S proteins often co-occur with T6SS components that are smaller in size and are largely uncharacterized (TagJ and TagB proteins).…”

“…Because of its central role, TssA is found in all T6SSs, nonetheless it is one of the least conserved core components of the apparatus (File S1; percentile identity of TssA proteins can be low even within the same phylogenetic group). Notably, TssA exists in two major forms, a long 55-60 kDa protein (TssA L ) and a shorter approximately 40 kDa version (TssA S ) (28, 29) (Fig. 1B).…”

“…The structure and function of prototypical TssA L proteins have been extensively studied (27–29) and it is known that the Escherichia coli , the Vibrio cholerae and the Aeromonas hydrophila TssA L form a ring-like structure, where the C-terminus of the protein largely occupies the center of the ring (27–29). It has been recently shown that these long TssA forms interact with an accessory TssA-like protein, called TagA, which secures the distal end of the sheath at the opposing cell membrane, ensuring optimal sheath polymerization and maintenance of the sheath in its extended conformation for long periods of time (29, 30). In contrast, Pseudomonas aeruginosa and Burkholderia cenocepacia TssA S , whilst still ring-like, display structures where the center of the ring is empty (26, 28).…”