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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 of Streptococcus 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.

SUBMITTER: Echelman DJ

PROVIDER: S-EPMC5448130 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Echelman Daniel J DJ Lee Alex Q AQ Fernández Julio M JM

The Journal of biological chemistry 20170327 21

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 exp ...[more]

PMID: 28348083

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Project description:Plasmodium falciparum (Pf) is responsible for the most lethal form of malaria. VAR2CSA is an adhesin protein expressed by this parasite at the membrane of infected erythrocytes for attachment to the placenta, leading to pregnancy-associated malaria. VAR2CSA is a large 355 kDa multidomain protein composed of nine extracellular domains, a transmembrane helix, and an intracellular domain. VAR2CSA binds to Chondroitin Sulphate A (CSA) of the proteoglycan matrix of the placenta. Shear flow, as the one occurring in blood, has been shown to enhance the (VAR2CSA-mediated) adhesion of Pf-infected erythrocytes on the CSA-matrix. However, the underlying molecular mechanism governing this enhancement has remained elusive. Here, we address this question by using equilibrium, force-probe, and docking-based molecular dynamics simulations. We subjected the VAR2CSA protein-CSA sugar complex to a force mimicking the tensile force exerted on this system due to the shear of the flowing blood. We show that upon this force exertion, VAR2CSA undergoes a large opening conformational transition before the CSA sugar chain dissociates from its main binding site. This preferential order of events is caused by the orientation of the molecule during elongation, as well as the strong electrostatic attraction of the sugar to the main protein binding site. Upon opening, two additional cryptic CSA binding sites get exposed and a functional dodecameric CSA molecule can be stably accommodated at these force-exposed positions. Thus, our results suggest that mechanical forces increase the avidity of VAR2CSA by turning it from a monovalent to a multivalent state. We propose this to be the molecular cause of the observed shear-enhanced adherence. Mechanical control of the valency of VAR2CSA is an intriguing hypothesis that can be tested experimentally and which is of relevance for the understanding of the malaria infection and for the development of anti placental-malaria vaccines targeting VAR2CSA.

Project description:The bacterial pathogen Staphylococcus pseudintermedius is involved in canine otitis externa and pyoderma as well as in surgical wound and urinary tract infections. Invasion of canine epithelial cells is promoted by S. pseudintermedius fibronectin (Fn)-binding proteins SpsD and SpsL through molecular interactions that are currently unknown. By means of single-molecule experiments, we discover that both adhesins have distinct molecular mechanisms for binding to Fn. We show that the SpsD-Fn interaction has a strength equivalent to that of a covalent bond (∼1.5 to 1.8 nN), which is an order of magnitude stronger than the binding force of classical receptor-ligand complexes. We suggest that this extreme mechanostability originates from the β-sheet organization of a tandem β-zipper. Upon binding to FnI modules, the intrinsically disordered binding sequences of SpsD would shift into an ordered structure by forming additional β-strands along triple peptide β-sheets in the Fn molecule. Dynamic force measurements reveal an unexpected behavior, i.e., that strong bonds are activated by mechanical tension as observed with catch bonds. By contrast, the SpsL-Fn interaction involves multiple weak bonds (∼0.2 nN) that rupture sequentially under force. Together with the recently described dock, lock, and latch complex, the ultrastrong interaction unraveled here is among the strongest noncovalent biological interaction measured to date. Our findings may find applications for the identification of inhibitory compounds to treat infections triggered by pathogens engaged in tandem β-zipper interactions.IMPORTANCE Binding of Staphylococcus pseudintermedius surface proteins SpsD and SpsL to fibronectin (Fn) plays a critical role in the invasion of canine epithelial cells. Here, we discover that both adhesins have different mechanisms for binding to Fn. The force required to separate SpsD from Fn is extremely strong, consistent with the unusual β-sheet organization of a high-affinity tandem β-zipper. By contrast, unbinding of the SpsL-Fn complex involves the sequential rupture of single weak bonds. Our findings may be of biological relevance as SpsD and SpsL are likely to play complementary roles during invasion. While the SpsD β-zipper supports strong bacterial adhesion and triggers invasion, the weak SpsL interaction would favor fast detachment, enabling the pathogen to colonize new sites.

Dataset Information

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

Publications

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

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