High Force Magnetic Tweezers Reveal That Bacterial Adhesion Pili Act as Megadalton-scale Schock Absorbers

Abstract

Gram positive pathogens colonize the oral cavity with their adhesive pili. These organelles are exposed to large mechanical perturbations, such as mastication or teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2nd and the 3rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2nd and 3rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to ∼4·105 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.