title: Mechanical forces regulate the reactivity of a thioester bond in a bacterial adhesin.
abstract: Bacteria must withstand large mechanical shear forces when adhering to and colonizing hosts. Recent structural studies on a class of Gram-positive bacterial adhesins have revealed an intramolecular Cys-Gln thioester bond that can react with surface-associated ligands to covalently anchor to host surfaces. Two other examples of such internal thioester bonds occur in certain anti-proteases and in the immune complement system, both of which react with the ligand only after the thioester bond is exposed by a proteolytic cleavage. We hypothesized that mechanical forces in bacterial adhesion could regulate thioester reactivity to ligand analogously to such proteolytic gating. Studying the pilus tip adhesin Spy0125 ofStreptococcus pyogenes, we developed a single molecule assay to unambiguously resolve the state of the thioester bond. We found that when Spy0125 was in a folded state, its thioester bond could be cleaved with the small-molecule nucleophiles methylamine and histamine, but when Spy0125 was mechanically unfolded and subjected to forces of 50-350 piconewtons, thioester cleavage was no longer observed. For folded Spy0125 without mechanical force exposure, thioester cleavage was in equilibrium with spontaneous thioester reformation, which occurred with a half-life of several minutes. Functionally, this equilibrium reactivity allows thioester-containing adhesins to sample potential substrates without irreversible cleavage and inactivation. We propose that such reversible thioester reactivity would circumvent potential soluble inhibitors, such as histamine released at sites of inflammation, and allow the bacterial adhesin to selectively associate with surface-bound ligands.
authors.affiliation: From the Department of Biological Sciences, Columbia University, New York, New York 10027 dje2122@columbia.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Echelman
authors.affiliation: From the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Alex Q
authors.initials: AQ
authors.lastname: Lee
authors.affiliation: From the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernández
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006632:Histamine / Q000737:chemistry*; D008744:Methylamines / Q000737:chemistry*; D017510:Protein Folding; D013297:Streptococcus pyogenes / Q000737:chemistry*
chemical_list: D018829:Adhesins, Bacterial; D004220:Disulfides; D008744:Methylamines; D006632:Histamine; C027451:methylamine
keywords: Gram-positive bacteria; adhesin; atomic force microscopy (AFM); bacterial adhesion; pilus mechanics; single-molecule biophysics; thioester bond