Structural insights into the function of type VI secretion system TssA subunits

Abstract

The type VI secretion system (T6SS) is a multi-protein complex that injects bacterial effector proteins into target cells. It is composed of a cell membrane complex anchored to a contractile bacteriophage tail-like apparatus consisting of a sharpened tube that is ejected by the contraction of a sheath against a baseplate. We present structural and biochemical studies on TssA subunits from two different T6SSs that reveal radically different quaternary structures in comparison to the dodecameric E. coli TssA that arise from differences in their C-terminal sequences. Despite this, the different TssAs retain equivalent interactions with other components of the complex and position their highly conserved N-terminal ImpA_N domain at the same radius from the centre of the sheath as a result of their distinct domain architectures, which includes additional spacer domains and highly mobile interdomain linkers. Together, these variations allow these distinct TssAs to perform a similar function in the complex.

Fig. 1

Phylogeny and domain organisation of TssA family proteins. a Proposed domain organisation of TssA1^A, TssA1^B, TssA2^A and TssA2^B subunits based on structural studies, secondary structure prediction and amino acid sequence alignment. The interdomain linkers comprise regions of variable length and amino acid sequence. The N-terminal conserved region, ImpA_N (comprised of three major regions of conservation, ImpA_N1-3), Nt1, Nt2, CTD and EX (C-terminal extension present in TssA2^B proteins) are indicated. Scale bar corresponds to 50 amino acids. b Maximum-likelihood phylogenetic tree from amino acid sequences of 47 TssA family members, showing the subdivision into three major clades as observed previously but with a revised nomenclature. The TssA2 clade is further divided into sub-clades A and B, based on patterns of sequence identity. TssA members discussed in detail in this study are highlighted in dark yellow. Scale bar represents 0.2 substitutions per site

Fig. 2

Interaction of TssA1^B with other T6SS subunits. a Two-hybrid analysis (maltose phenotypes). Hybrid proteins are represented by a green or yellow coloured motif representing the CyaA fragment (T25 and T18, respectively) linked to a white rectangle labelled according to the fused T6SS subunit, as shown in the key at the bottom of (b). T6SS subunits are indicated by a single letter corresponding to the suffix used in the Tss nomenclature (i.e. A1 corresponds to TssA1^B, B corresponds to TssB etc) except for H (Hcp), V (VgrG) and V_C (VgrG core region). M_N and M_C represent the N-terminal cytoplasmic and C-terminal periplasmic regions of TssM, respectively. The efficiency of complementation of representative combinations (phenotypes shown in red font) were determined by β-galactosidase assay. b Two-hybrid analysis (β-galactosidase activity). Data is representative of three independent experiments (n = 3) performed in duplicate and values correspond to the mean ± standard deviation. Nomenclature as in (a). Z represents the Zip control. Values are presented in Supplementary Data 1. c Co-immunoprecipitation analysis. FLAG-tagged Bc TssA1^B and potential interacting T6SS subunits (TssX) tagged with the VSV-g or HA epitope tags, or with MBP, were co-expressed in E. coli. FLAG.TssA1^B was immunoprecipitated from cell lysates and recovered prey proteins (IP) were detected with the appropriate epitope antibody by western blotting. MBP was included as a control and was detected with MBP pAb. Proteins present in the cell lysate (Tot) and the unbound material (UB) were also analysed. Blots were also probed with FLAG mAb (an example of such a blot following co-expression of FLAG.TssA1^B and VSVg.TssM_N is shown in the bottom panel). Uncropped images of the blots are shown in Supplementary Fig. 4. d Bc TssA1^B interaction network. Interactions between Bc TssA1^B and other T6SS subunits are indicated by red arrows. Previously reported interactions are indicated by grey connectors. Note that VgrG and TssK have been shown to interact with the TssF-TssG complex but for simplicity are linked to TssF in this figure

Fig. 3

Effect of TssA1^B inactivation on T6SS secretion activity. Cell associated protein (CA) and spent supernatants (SN) from cultures of B. cenocepacia H111 (WT) and the isogenic tssA1^B mutant (tssA::Tp), each with or without pBBR1MCS (VC) or pBBR1MCS-tssA1^B (ptssA), were fractionated by SDS-PAGE, blotted onto PVDF membrane and probed with a Hcp pAb or a mAb specific for the RNAP β subunit (lysis control). Detection of bound antibodies was with HRP-conjugated secondary antibodies. The tssM mutant (ΔM) was included as a control as TssM has been shown to be an essential component of the B. cenocepacia T6SS. Uncropped images of the blots are shown in Supplementary Fig. 5

Fig. 4

High-resolution structure of Bc TssA1^B Nt1 and its similarity to Ah TssA2 Nt2. a Bc TssA1^B Nt1 sequence annotated to show the elements of secondary structure. Location of α-helices are shown by coloured rectangles above the amino acid sequence with the region corresponding to Sd1 highlighted in yellow and Sd2 highlighted in red. The three ImpA_N motifs are indicated in orange font and conserved ImpA_N domain residues (see Supplementary Fig. 1) are shown in black boxes. b X-ray structure of Bc TssA1^B Nt1 (PDB: 6HS5 https://www.ebi.ac.uk/pdbe/entry/pdb/6hs5). The two subdomains, Sd1 and Sd2, are shown in yellow and red, respectively. c X-ray structure of Bc TssA1^B Nt1 represented as in (b) showing location of ImpA_N1-ImpA_N3 regions in orange. For clarity, the helices of Sd2 (α6-α11) located C-terminal to ImpA_N3 are made more transparent than those in Sd1. d Bc TssA1^B Sd1 (orange) superimposed on the Ah TssA2^A Nt2 domain (PDB: 6G7B https://www.ebi.ac.uk/pdbe/entry/pdb/6g7b; cyan) showing the close structural similarity of helices α1-α5 of Bc TssA1^B Nt1 (majority of ImpA_N) to helices α2-α6 of Ah TssA2^A Nt2. α-helices are shown as cylinders

Fig. 5

Structure of the Bc TssA1^B CTD ring. a Negative stain EM of TssA1^B particles. The magnified view indicates the location of the Nt1 domains (arrows) presented around the CTD oligomeric ring (circle). Scale bar corresponds to 50 nm. b Bc TssA1^B CTD monomers assemble to form a double-layered ring containing 32 subunits exhibiting D₁₆ symmetry (PDB: 6HS6 [https://www.ebi.ac.uk/pdbe/entry/pdb/6hs6]). The views shown correspond to a top view down the 16-fold axis and a side view perpendicular to the 16-fold axis, with the dimensions of the ring shown. c, d Dimerisation of Bc TssA1^B CTD monomers. c Side view from inside the Bc TssA1^B CTD ring showing interaction of residues from helices α12, α13 and α14 within a dimer (chain A, purple; chain B, cyan). Interacting surfaces are highlighted (chain A, light pink; chain B, blue). d Formation of the salt bridge between R306 and E324 that caps both ends of the dimer interface. e, f Interactions between dimers to form the Bc TssA1^B CTD ring. e Expanded view of the two chains shown in purple and orange in (b), to show interactions leading to ring assembly. f Side view from inside the Bc TssA1^B CTD ring showing the interlocking interaction between helices α13, α14 and α15 from two dimers which are critical to ring formation around the 16-fold axis: dimer 1, purple/cyan; dimer 2, blue/orange. The interfacing regions of the purple and orange monomers are shown in light pink and yellow, respectively. g Structure-based sequence alignment of the Pa TssA1^A and Bc TssA1^B CTD domains. The secondary structure of TssA1^B CTD is highlighted above the sequence

Fig. 6

Structure of the Ah TssA2^A CTD ring. a Negative stain EM particle averaging of Ah TssA2^A CTD indicating five-fold symmetry. b Structure of 10 Ah TssA2^A CTD monomers assembled to form a decameric oligomer exhibiting D₅ symmetry (PDB: 6G7C https://www.ebi.ac.uk/pdbe/entry/pdb/6g7c). Boxed region 1 (Interface 1), two-fold axis formed by dimerisation via the WEP motif (red and blue chains). Boxed region 2 (Interface 2), two-fold axis generated through packing of helices α9, α10 and α11 (blue and orange chains). The N-terminus of chain A is indicated and the dimensions of the ring are shown. c Interface 1 with key interacting residues indicated (chain A, pink: chain B, light blue). d Side view of interface 2 with interacting surfaces highlighted in light blue and sand. e Superposition of WEP-mediated dimers from Ah TssA2^A (cyan) and EAEC TssA2^B (PDB: 4YO5, green) about the WEP interface. The monomers exhibit a different relative orientation of ~35° about the WEP interface. View corresponds to chains A and B shown in (b). f Structural similarity of the CTD monomers of Ah TssA2^A (cyan) and EAEC TssA2^B (PDB: 4YO5, green). Helices α13 and α14 are unique to EAEC TssA2^B and form the C-terminal extension (magenta), which is important for the different quaternary structures assembled by TssA2^A and TssA2^B. g, h Side by side views of the Ah TssA2^A (g) and EAEC TssA2^B (h) CTD oligomers with their respective five-fold and six-fold axes vertical. Assembly of Ah TssA2^A WEP-mediated dimers (cyan) around the five-fold axis involves a rotation relative to EAEC TssA2^B (green) of ~50° around the dimer axis as illustrated by the dotted black line. The neighbouring subunits with which each monomer within a WEP-mediated dimer interacts are shown in grey

Fig. 7

Schematic representation of TssA ring dimensions and domain flexibility. a Arrangement of the Ah TssA2^A Nt2 domains around the CTD oligomer as observed in the asymmetric unit. The Nt2 domains do not follow strict five-fold symmetry. The decameric CTD ring is shown in dark grey perpendicular to the five-fold (left) and two-fold (right) axes with each peripheral Nt2 dimer shown in a different colour. The diagram between the two structures shows the relationship between dimer-forming Nt2 domains (represented by coloured squares) and their corresponding C-terminal domains in the TssA2^A Nt2-CTD decamer. b Displacement of the Ah TssA2^A Nt2 dimers perpendicular to the five-fold axis of the CTD oligomer. The CTD ring is shown in light grey with one WEP-mediated CTD dimer indicated in cyan to which is tethered an Nt2 dimer. The Nt2 dimer (light green) is shown at its maximum displacement of ~60° either side of the plane of the CTD ring (orange line). Other observed positions of Nt2 are indicated by translucent shading. c Top and side view representations showing the domain architecture of TssA subunits from clades 1^B, 2^A and 2^B modelled on the baseplate-distal end of the T6SS tube/sheath assembly during polymerisation. The V. cholerae TssBC extended sheath (green) surrounding a Hcp hexamer (yellow) (PDB: 5MXN) is shown on the left. Onto this, is modelled the TssA1^B CTD oligomer (orange) with 16-fold symmetry, showing one stacked CTD dimer attached to a pair of Nt1 domains via flexible linkers, the TssA2^A CTD oligomer (blue) with five-fold symmetry, showing a WEP-mediated CTD dimer connected to the two monomers of an Nt2 dimer via flexible linkers, each of which are in turn connected to an Nt1 domain, and the TssA2^B CTD oligomer (red) displaying six-fold symmetry, showing a CTD dimer connected to the two monomers of an Nt2 dimer via flexible linkers, each of which are in turn connected to an Nt1 domain. Labels are: C, CTD dimers (top view); N1, Nt1; N2, Nt2; CTD, CTD oligomer (side view). Scale bar corresponds to 65 Å