Impact of an alpha helix and a cysteine–cysteine disulfide bond on the resistance of bacterial adhesion pili to stress

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking β-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

Escherichia coli have a remarkable ability to adapt to the environment, allowing these bacteria to colonize varying niches in humans and animals either as commensals or pathogens (1). The urinary and gastrointestinal tracts are examples of environments where pathogenic E. coli are common causes of acute urinary tract infections and severe diarrhea, respectively. In these niches, fluid flow is a natural defense mechanism, limiting attachment of pathogenic bacteria to epithelial cell surfaces (2, 3). To facilitate attachment under fluid flow that applies drag forces to a bacterium, E. coli use attachment organelles called adhesion pili or fimbriae (4) that are micrometer-long helical rod structures (Fig. 1A). The helical rod structure can unwind (5), and significantly extend its original length under tensile force. This unwinding allows cell-associated bacteria to withstand drag forces from fluid flow, by decreasing the load on the receptor-bound adhesin (6, 7) that binds to host receptors (8). The unwinding of pili is dependent on critical mechanical features of the fibers. If the pili are compromised, the bacteria’s ability to attach and stay attached under drag force is reduced significantly (9, 10), and enterotoxigenic E. coli (ETEC) with no pili are unable to cause disease (11). Uropathogenic E. coli (UPEC) and ETEC pili mechanics and structure have been investigated for decades, yet we still lack a complete picture of their mechanical differences and how these differences relate to their genetics, structure, and environmental niche.Open in a separate windowFig. 1.(A) Scanning electron microscopy image of E. coli bacteria expressing adhesion pili. (B) Cartoon of a pilus showing subunits assembled into a helix-like rod stabilized by layer-to-layer interactions between subunits n and n ± 3, and a region of unwinding. (C) High-resolution structure of a P pilus with each pilin subunit colored individually. (D) A trimer (3mer) of pilin subunits during unwinding. (PapA PDB ID: 5FLU.)Pili that are assembled via the “chaperone–usher pathway” are all genetically similar. These pili are “class 1 adhesion pili,” and pili from this class are expressed on both UPEC and ETEC bacteria, comprising class 1a and class 1b, respectively (12). Conversely, ETEC adhesion pili from the ‘alternate chaperone–usher pathway’ do not share this genetic similarity, comprising class 5 pilins (13). Structurally, both pilus types are composed of immunoglobulin (Ig)-like pilin subunits attached via a β-strand complementation head to tail, forming helical fibers of approximately 1,000 pilins with similar quaternary structure (14, 15). The stability of the quaternary helical rod structure is achieved via layer-to-layer bonds formed between subunits, primarily between subunits n and n + 3 (Fig. 1B). High-resolution three-dimensional helical reconstruction models exist for UPEC class 1 type 1 and P pili (Fig. 1 C and D), whereas the only class 5 ETEC pilus that has been reconstructed at a resolution sufficient for atomic model building is CFA/I pili (10, 16, 17). These reconstructions show that P pili and type 1 pili have a larger buried surface area between subunits n and n + 3 than CFA/I pili (1,616 Ų and 1,453 Ų, respectively, vs. 1,087 Ų). The magnitude of the buried surface area correlates well with the force needed to unwind the fibers. That is, larger buried surface area requires a higher tensile force: The force needed to unwind type 1 and P pili is more than fourfold that of CFA/I pili, ≈30 and 28 pN vs. 7 pN (17, 18). Overall, the unwinding capabilities of pili are well understood, and good biophysical models explain the measured force–extension curves (19–22). However, current force–extension data indicate that there is a puzzling difference in the mechanics of pili that these models cannot explain, that could be related to pilin sequence differences. Class 1 pili show an additional conformational change that takes place at almost twice the pilus unwinding force (≈60 pN), which makes them more elastic and allows significantly longer extensions than those of previously studied class 5 pili (18, 23–25). Since the conformational change in class 1 pili takes place after unwinding of the quaternary structure, it must occur when a pilus is already in its linearized form (subunits in a head-to-tail order). That is, after unwinding the helical rod, there are changes in secondary or tertiary structures of individual pilin subunits. However, these changes have not been explored.It is well established that class 1 pilin subunits are proteins with high mechanical stability. Pilin stability originates from the Ig-like structure that is assembled of six β-strands forming a β-sandwich, and includes a conserved disulfide bond that works as a mechanical lock (26, 27). These physical attributes yield pilins that are characterized by very high thermodynamic and kinetic stability, free energies of over 70 kJ/mol, and a half-life of 10⁸ y at 25 °C (26, 28), as well as being robust under tensile force (27). In contrast, little is known regarding the physical attributes of class 5 pilin stability, except that there are striking similarities with class 1 pilins regarding their IgG protein fold and size (25). Thus, we raise the following question: What provides the mechanical differences observed between class 1 and class 5 adhesion pili, and are differences related to environmental niche?To solve the aforementioned research question, we compared the mechanical differences and structural properties of UPEC- and ETEC-associated pili. We measured their mechanical properties using optical tweezers (OT) force spectroscopy, and we interpreted the experimental force–extension results in the final region of pilus extension using structural models and steered molecular dynamics (sMD) simulations. We also looked closely at class 1 ETEC pili that have more genetic homology to UPEC pili as compared to class 5 ETEC, to examine the relation between genetics, pilus mechanics, and environmental niche.