Surviving a Bumpy Ride in the Oropharynx: Bacterial Pili as Nano-Seatbelts that Dissipate Mechanical Energy

Abstract

Bacterial pili function in cellular adhesion, and must withstand large mechanical stresses in host environments, such as coughing and chewing. In gram positive bacteria, pili are covalently-linked polymers of single protein subunits, termed pilins. Gram positive pilins uniquely possess intramolecular isopeptide bonds that bridge the peptide backbone to form bypass force transduction pathways. In the crystal structure of Spy0128, a pilin from Streptococcus pyogenes, isopeptide bonds link the N- and C-terminal β-strands. Consequently, Spy0128 is mechanically inextensible. Here we report on the mechanical properties of two related pilins, SpaA from Corynebacterium diphtheriae and FimA from Actinomyces oris, using atomic force microscopy (AFM)-based single molecule force spectroscopy. In the crystal structures of SpaA and FimA, the isopeptide bonds do not directly link the N- and C-terminal β-strands in a single pilin domain. Instead, the isopeptide arrangement creates a ∼40 residue polypeptide loop that resembles a slackened seatbelt, which we predict is sensitive to mechanical unfolding. We find that both SpaA and FimA extend to 14 nm under mechanical force, consistent with our structure-based prediction of unfolding of the nano-seatbelt from a slackened to a taut conformation. At a loading rate of 400 nm/s, these loops unfold at forces of ∼503pN in SpaA and ∼665pN in FimA; as such, SpaA and FimA are among the most mechanically stable proteins yet reported. When the force perturbation is removed, the loops refold at a rapid rate of 29 s−1 or higher. Remarkably, the mechanical stabilities are ∼75pN weaker upon refolding, suggesting that gaining full mechanical stability requires maturation. The high mechanical stability and rapid refolding of the nano-seatbelts suggest a mechanism whereby pilin subunits, polymerized as tens-to-hundreds of repeats in pili, readily absorb and recover from mechanical shocks.