Archaic chaperone–usher pili self-secrete into superelastic zigzag springs

Abstract

Adhesive pili assembled through the chaperone–usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1–3. Archaic chaperone–usher pathway pili, the most diverse and widespread chaperone–usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly–secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii—a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.

Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa

“…While the subunit arrangement is overall similar in the CupE pilus, there are significantly fewer interacting residues between subsequent subunits in the CupE pilus compared to the Csu pilus, despite the high structural and sequence similarity between the pilins (RMSD = 1.55 Å; S4 and S5 Figs). Thus, the CupE pilus shows a relaxed arrangement of subunits compared to the tight packing of the Csu pilus, resulting in a higher rise per subunit than in the Csu pilus (S5B Fig) . The relaxed subunit arrangement suggests the possibility that the CupE pilus may be more flexible than the Csu pilus, which has been described as laterally stiff [40]. Indeed, increased lateral curvature within CupE pili was observed in a subset of two-dimensional class averages (S3B Fig), supporting some degree of flexibility around the subunit-subunit interface that allows the orientation of subunits to vary.…”

“…The interface between the Ig-like domains is relatively small in comparison to the extensive donor-strand interactions, and is mainly maintained by the interaction of a loop with the next subunit, with two interacting valine residues V69 and V48 at the core of the interaction (Fig 2D). In a recent publication on the related Csu pilus, interactions between zigzag-arranged subunits within an archaic CUP pilus were termed 'clinch' interactions [40]. While the subunit arrangement is overall similar in the CupE pilus, there are significantly fewer interacting residues between subsequent subunits in the CupE pilus compared to the Csu pilus, despite the high structural and sequence similarity between the pilins (RMSD = 1.55 Å; S4 and S5 Figs).…”

“…stiff, Csu pilus from A. baumannii can be explained by the number of interacting residues at the inter-subunit interface: The Csu pilus, which was characterised in a recent study by overexpression of the corresponding csu operon in E. coli [40] has increased subunit interactions within the pilus as a result of denser packing compared with CupE (S5 Fig).…”

“…Interestingly, it was found in optical tweezer experiments that the Csu pilus can be extended to almost twice its length along the helical axis by breaking of the subunit interface contacts [40]. Given that CupE shares a similar architecture, it seems likely that it shares this super-elasticity, suggesting it may be a common feature of archaic CUP pili.…”

Structure of thePseudomonas aeruginosaPAO1 Type IV pilus

“…The N-terminal a-helix of PilA extends away from the body of the pilin globular domain to stack with other pilin subunits, mediating major subunit-subunit interactions within the pilus. Such a 'hydrophobic arm'-like element is also commonly found in other pili where it similarly provides extensive contact with hydrophobic regions of other subunits, for example in filaments employing donor-strand exchange 53,58,59 . A unique feature of T4Ps is the simultaneous interaction of one N-terminal region with various regions of several other subunits within the pilus (Figs.…”