CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Abstract

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.bacterial adhesion | mechanical stability | single-molecule force spectroscopy | Gram-positive pili | isopeptide bond B acterial infections of solid tissues begin with the attachment of bacteria to target surfaces. In many instances, bacteria adhere against forces that oppose such attachment: micturition in the genitourinary tract (1) or mucociliary flow in the respiratory tract (2), for example. In such environments, a completely immobile adherent bacterium experiences a drag force that can be approximated by Stokes law, F = 6·π·r·η·v, where r is the Stokes radius of the bacterium (∼0.5 μm), η is the viscosity of the fluid (in the respiratory mucus, 1-100 Pa·s −1 ) (3), and v is the velocity of the fluid surrounding the bacterium (Fig. 1A). Under normal mucociliary flow (1-100 μm·s −1 ) (4), forces on a single bacterium can exceed several nanonewtons. Such high forces are sufficient to cleave covalent bonds within the initial adherence structures (5), which would terminate attachment. Understanding how bacteria manage to remain attached under such strong mechanical perturbations is of fundamental interest and could identify new targets for antibiotic development.The initial interaction between bacteria and the host is mediated by micrometer-long adhesive structures termed pili or fimbriae (Fig. 1A). Due to their adhesive role, pili are virulence factors that contribute to the development of infections (6). Structurally, pili are polymers of tens to hundreds of subunits, termed shaft pilins, that are assembled in series and are presented at the extracellular surface, often with inclusion of minor pilins that can have adhesive properties (6). Remar...

Proteins Breaking Bad: A Free Energy Perspective

“…The discovery that massively large tandem modular proteins play a crucial role in cellular functioning has shifted our view on the molecular origins and regulation of protein mechanics 1–6 . For instance, the unfolding/refolding of the numerous domains of the giant protein titin contributes to the elasticity of the muscle, and assists muscle contraction 5 .…”

Mechanical forces regulate the reactivity of a thioester bond in a bacterial adhesin

“…These internal covalent bonds have a pronounced effect on the mechanical properties of their proteins, often increasing stability, accelerating folding, and delimiting extensibility (16,19).…”

“…For CnaB-TED, we predict an unfolding extension of 119 nm (Table 1). However, an internal thioester bond could protect those residues sequestered behind the bond from mechanical unfolding, similar to the action of internal disulfide and isopeptide bonds (16,19). Consequently, the predicted extension is proportionately reduced by the number of residues sequestered behind the bond.…”

“…S2). When the thioester bond is present, 1 nm is added based on the estimated extended length of an internal isopeptide bond (16). In CnaB D595A alone, 1.1 nm is added for the measured length of the TED mechanical clamp between Ala-393 and Gly-579 (supplemental Fig.…”

“…Moreover, mechanical unfolding exposes buried residues to chemical modification (14,15). Bacterial adhesins can experience extreme physiological shearing forces, and certain Gram-positive adhesins are correspondingly adapted to remain folded up to ϳ700 pN, the highest forces reported for protein mechanical stability (16). Could mechanical force also fine-tune the reactivity of the thioester bond in bacterial adhesins?…”