PilY1 regulates the dynamic architecture of the type IV pilus machine in Pseudomonas aeruginosa

Abstract

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics.

Fig. 1. The in-situ architecture of the P. aeruginosa T4P machine reveals a cage-like structure that spans the cell envelope.

a Slice through a tomogram of the cell pole of a wild-type P. aeruginosa (similar results were observed in >100 tomograms). b Central slice of the subtomogram-averaged structure of the piliated T4P machine from the ΔpilT cells of P. aeruginosa. c Central slice of the subtomogram-averaged structure of the non-piliated T4P machine from the ΔpilB cells of P. aeruginosa. d Subtomogram-averaged structures of the piliated (14) and non-piliated (64) Pa-T4P machines are mapped back into the segmented bacterial membranes of the tomogram shown in (a). Also see Supplementary Movie 1. e In-situ architectures of the piliated ΔpilT and non-piliated ΔpilB Pa-T4P machines assembled from segmented focus-refined maps (f, Fig. 4g; Supplementary Fig. 8a, b) of the nanomachine. OM outer membrane, PG peptidoglycan, IM inner membrane. f Central slice of the focus-refined subtomogram-averaged structure of the upper and mid-cage regions of the piliated ΔpilT T4P machine. g Cryo-EM single-particle structure of P. aeruginosa secretin PilQ_N1-secretin (PDB: 6ve2/EMDB ID: EMD-21152, secretin gate opened to accommodate the pilus) in complex with TsaP_CI-II (pink) fitted into the subtomogram-averaged map of the upper and mid-cage of the T4P machine. h Top view of the upper cage.

Fig. 2. Structural details of the secretin and its interface with the alignment subcomplex.

a Central slice of the focus-refined subtomogram-averaged structure of the upper and mid-cage regions of the T4P machine from ΔtsaP cells. b Structure of PilQ (yellow for PilQ_N1-secretin solved by cryo-EM (PDB:6VE3/EMDB ID:EMD-21153) and orange for PilQ_Amin2 and PilQ_N0 structures predicted by AlphaFold) fitted into the subtomogram-averaged map of the Pa-T4P machine from ΔtsaP cells. c Structure of PilQ (yellow for PilQ_N1-secretin solved by cryo-EM and orange for the PilQ_Amin1,2-N0 structure predicted by AlphaFold) in complex with TsaP (pink for TsaP_CI-II solved by cryo-EM and magenta for TsaP_LysM predicted by AlphaFold) and PilP_C fitted into the segmented subtomogram-averaged map of piliated Pa-T4P machines from ΔpilT cells. d Cross-section view of the Pa-T4P machine derived from the hyperpiliated ΔpilT cells showing its extended shape that contacts PG. e Pseudoatomic structure of the PilQ-TsaP-PilP_C complex anchored within the segmented cryo-ET density of OM and PG in the piliated ΔpilT Pa-T4P machine. f Surface representation view of the bottom of the PilQ-TsaP-PilP_C complex in the piliated ΔpilT Pa-T4P machine.

Fig. 3. In-situ architecture of the P. aeruginosa T4P machine.

a Segmented focus-refined map (raw density map in Supplementary Fig. 8a) of the lower and cytoplasmic cage regions of the piliated T4P machine. b AlphaFold-Multimer-predicted structure of the PilP (light green)—PilO (yellow; transmembrane region, red)—PilN (blue; transmembrane region, red)—PilM (dark green) complex fitted into the segmented cryo-ET map (transparent). c Architecture of the c7-symmetric alignment subcomplex fitted into the subtomogram-averaged cryo-ET map. The cryo-ET maps are transparent, with the structures docked in their interiors. d Cross-section view of the lower periplasmic cage that shows the c7-symmetry complex PilP-PilO-PilN surrounding the pilus in the center. The relative position of (d) is shown with an arrow in (a). The spacing between two neighboring pillars (composed of PilP-N-O) is ~38 Å, which enables the free diffusion of major pilin PilA to enter and exit the T4P machine within the inner membrane. e PilC trimer model fitted into the segmented cryo-ET density. f Cross-section view of the T4P machine showing the AlphaFold-predicted PilC trimer and the cryo-EM structure of the P. aeruginosa PAO1 T4P filament (PDB: 8TUM) fitted into the segmented cryo-ET maps. The motor (light blue) is illustrated with the cryo-ET density. g In-situ architectural model of the piliated T4P machine of P. aeruginosa.

Fig. 4. Proposed role of PilY1 in regulating the dynamic T4P architectures.

a A central slice of a tomogram of ΔpilY1 cells (similar results were observed in >100 tomograms). Yellow boxes indicate some of the T4P machines seen in the tomogram. b–e 3-D classification of the subtomogram-averaged structure of the ΔpilY1 Pa-T4P machine. The percentage of the particles and spacing of the periplasm are indicated. f Central slice of the focus-refined subtomogram-averaged structure of the top region of the ΔpilY1 T4P machine. g Central slice of the focus-refined subtomogram-averaged structure of the top region of the non-piliated ΔpilB T4P machine. h Structure of PilY1 predicted by AlphaFold2. i AlphaFold predicted structure of PilQ in complex with TsaP, PilP_C, and PilY1 fitted into the focus-refined subtomogram-averaged maps of the ΔpilB T4P machine. j A zoomed-in view of (i) where PilY1 interacts with PilQ and the minor pilin complex. Insert domain, orange; vWA domain, magenta; β-propeller domain, red.

Fig. 5. Proposed model of the dynamic assembly of the Pa-T4P machine.

a Compared to those in the planktonic state, surface-attached P. aeruginosa overproduce T4P leading to formation of biofilms. Parts of (a) were created in BioRender. Guo, S. (2024) BioRender.com/n16p426. b In the absence of PilY1, the assembly of the minor pilin and the alignment subcomplexes is suboptimal. c PilY1 and the minor pilin complex help optimize the conformations of the T4P machine for pilus dynamics. d, e PilY1 assembles to the pilus tip via the minor-pilin complex during the dynamic cycles of T4P extension and retraction.