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Julio M. Fernandez - One of the best experts on this subject based on the ideXlab platform.
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Mechanical forces regulate the reactivity of a thioester bond in a Bacterial Adhesin
The Journal of biological chemistry, 2017Co-Authors: Daniel J. Echelman, Alex Q. Lee, Julio M. FernandezAbstract: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.
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Force Spectroscopy of a Bacterial Adhesin with an Internal Thioester Bond
Biophysical Journal, 2016Co-Authors: Daniel J. Echelman, Julio M. FernandezAbstract:Thioester bonds are ubiquitous in biology, often serving as reactive intermediates, for example in proteolysis and fatty acid synthesis. In addition, stable thioester bonds between Cys and Gln side chains are found internally within immune complement proteins and anti-proteases, which utilize these bonds as potent substrates for covalent attachment to ligand. In these instances, exposure of the bond is said to be ‘cryptic’, requiring a proteolytic cleavage to unmask the buried thioester. Recently, thioester bonds have been discovered in Adhesin domains of Gram-positive bacteria. These Adhesins mediate the covalent attachment of bacteria to hosts, microbial biofilms, and surfaces, and consequently must sustain high levels of mechanical stress. Here we report the first mechanical characterization of a thioester-containing protein from studying the Streptococcus pyogenes Spy0125 Adhesin. We constructed a fusion protein of the three C-terminal domains of Spy0125 with an N-terminal HaloTag for bioorthogonal covalent anchoring, and further introduced two isopeptide knockout mutations into Spy0125 to provide an unambiguous fingerprint from mechanical polyprotein unfolding. Using AFM-based single-molecule force spectroscopy, we observe unfolding extensions of 15 nm within the Adhesin's thioester domain (TED), consistent with a single thioester bond delimiting extension. This thioester domain is inserted within a separate “host” domain in the crystal structure. Uniquely, unfolding follows a reverse mechanical hierarchy as the more mechanically stable host domain unfolds first, at a force of 288 ± 25 pN, followed by the weaker TED, at a force of 121 ± 14 pN, thereby protecting the TED and thioester bond. Both domains are observed to refold on timescales of under 1 second. We propose that reactivity of the thioester bond may be mechanically ‘cryptic’, sequestered within the TED and unmasked for covalent attachment only when the host domain is under force.
Peter L Davies - One of the best experts on this subject based on the ideXlab platform.
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structural basis of ligand selectivity by a Bacterial Adhesin lectin involved in multi species biofilm formation
bioRxiv, 2020Co-Authors: Shuaiqi Guo, Tyler D R Vance, H Zahiri, R Eves, C Stevens, Janhendrik Hehemann, S Vidalmelgosa, Peter L DaviesAbstract:Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 lectin is a domain of a 1.5-MDa Adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here we show MpPA14 binds various monosaccharides, with L-fucose and N-acetyl glucosamine being the strongest ligands (Kd ~ 150 uM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin9s apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3, 4 diols of L-fucopyranose and glucopyranoses, and binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end-groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by L-fucose, but not by N-acetyl galactosamine. With MpPA14 lectin homologs present in Adhesins of several pathogens, our work gives insight into an anti-adhesion strategy to block infection via ligand-based antagonists.
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Correction: Structure and functional analysis of a Bacterial Adhesin sugar-binding domain.
PloS one, 2019Co-Authors: Tyler D R Vance, Shuaiqi Guo, Brigid Conroy, Shayan Assaie-ardakany, Peter L DaviesAbstract:[This corrects the article DOI: 10.1371/journal.pone.0220045.].
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structure and functional analysis of a Bacterial Adhesin sugar binding domain
PLOS ONE, 2019Co-Authors: Tyler D R Vance, Shuaiqi Guo, Shayan Assaieardakany, Brigid Conroy, Peter L DaviesAbstract:Bacterial Adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX Adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa Adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-A resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar’s two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein’s ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-μM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.
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Structure of a 1.5-MDa Bacterial Adhesin reveals its role in the mixed-species biofilm formation with diatoms on ice
Acta Crystallographica Section A Foundations and Advances, 2018Co-Authors: Shuaiqi Guo, Ilja K. Voets, Peter L DaviesAbstract:Bacterial Adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa Adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6 μm-long Adhesin using a ‘dissect and build’ structural biology approach and have established complementary roles for its five distinct Regions. Domains in Region I (RI) tether the Adhesin to the Type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical Ig-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligandbinding domains that join diatoms and bacteria together in a mixed species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain; and the terminal RV domain contains several ‘Repeats-in-Toxin’ motifs and a non-cleavable signal sequence that target proteins for export via the Type I secretion system. Similar structural architecture is present in the Adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity
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Unusually high mechanical stability of Bacterial Adhesin extender domains having calcium clamps.
PloS one, 2017Co-Authors: As Anneloes Oude Vrielink, Tyler D R Vance, Peter L Davies, Arthur M. De Jong, Ilja K. VoetsAbstract:To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two Bacterial Adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 μm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other β-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.
Daniel J. Echelman - One of the best experts on this subject based on the ideXlab platform.
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Mechanical forces regulate the reactivity of a thioester bond in a Bacterial Adhesin
The Journal of biological chemistry, 2017Co-Authors: Daniel J. Echelman, Alex Q. Lee, Julio M. FernandezAbstract: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.
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Force Spectroscopy of a Bacterial Adhesin with an Internal Thioester Bond
Biophysical Journal, 2016Co-Authors: Daniel J. Echelman, Julio M. FernandezAbstract:Thioester bonds are ubiquitous in biology, often serving as reactive intermediates, for example in proteolysis and fatty acid synthesis. In addition, stable thioester bonds between Cys and Gln side chains are found internally within immune complement proteins and anti-proteases, which utilize these bonds as potent substrates for covalent attachment to ligand. In these instances, exposure of the bond is said to be ‘cryptic’, requiring a proteolytic cleavage to unmask the buried thioester. Recently, thioester bonds have been discovered in Adhesin domains of Gram-positive bacteria. These Adhesins mediate the covalent attachment of bacteria to hosts, microbial biofilms, and surfaces, and consequently must sustain high levels of mechanical stress. Here we report the first mechanical characterization of a thioester-containing protein from studying the Streptococcus pyogenes Spy0125 Adhesin. We constructed a fusion protein of the three C-terminal domains of Spy0125 with an N-terminal HaloTag for bioorthogonal covalent anchoring, and further introduced two isopeptide knockout mutations into Spy0125 to provide an unambiguous fingerprint from mechanical polyprotein unfolding. Using AFM-based single-molecule force spectroscopy, we observe unfolding extensions of 15 nm within the Adhesin's thioester domain (TED), consistent with a single thioester bond delimiting extension. This thioester domain is inserted within a separate “host” domain in the crystal structure. Uniquely, unfolding follows a reverse mechanical hierarchy as the more mechanically stable host domain unfolds first, at a force of 288 ± 25 pN, followed by the weaker TED, at a force of 121 ± 14 pN, thereby protecting the TED and thioester bond. Both domains are observed to refold on timescales of under 1 second. We propose that reactivity of the thioester bond may be mechanically ‘cryptic’, sequestered within the TED and unmasked for covalent attachment only when the host domain is under force.
David L. Hasty - One of the best experts on this subject based on the ideXlab platform.
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The FimH protein of type 1 fimbriae. An adaptable Adhesin.
Advances in experimental medicine and biology, 1996Co-Authors: Per Klemm, Mark A. Schembri, David L. HastyAbstract:The most common of the enteroBacterial adhesive surface organelles, and one of the best charachterized, is type 1 fimbriae. Although the exact biological role of these organelles has been somewhat controversial, recent evidence strongly indicates that type 1 fimbriae, among other things, are implicated in enhancing the virulence of certain uropathogenic strains. A detailed dissection of the molecular biology of type 1 fimbriae and their role in Bacterial Adhesin would undoubtedly help to reach a clearer understanding of important aspects of Bacterial pathogenesis.
Per Klemm - One of the best experts on this subject based on the ideXlab platform.
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the tiba Adhesin invasin from enterotoxigenic escherichia coli is self recognizing and induces Bacterial aggregation and biofilm formation
Infection and Immunity, 2005Co-Authors: Orla Sherlock, Rebecca Munk Vejborg, Per KlemmAbstract:Escherichia coli strains are responsible for many cases of gastrointestinal disease and represent a serious health problem worldwide. An essential step in the pathogenesis of such strains involves recognition and attachment to host intestinal surfaces. TibA is a potent Bacterial Adhesin associated with a number of enterotoxigenic E. coli strains and mediates Bacterial attachment to a variety of human cells; additionally, it promotes invasion of such cells. This Adhesin is a surface-displayed autotransporter protein and belongs to the exclusive group of Bacterial glycoproteins; only the glycosylated form confers binding to and invasion of mammalian cells. Here we characterized TibA and showed that it possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrated that intercellular TibA-TibA interaction is responsible for Bacterial autoaggregation. Also, TibA expression significantly enhances biofilm formation by E. coli on abiotic surfaces.
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The FimH protein of type 1 fimbriae. An adaptable Adhesin.
Advances in experimental medicine and biology, 1996Co-Authors: Per Klemm, Mark A. Schembri, David L. HastyAbstract:The most common of the enteroBacterial adhesive surface organelles, and one of the best charachterized, is type 1 fimbriae. Although the exact biological role of these organelles has been somewhat controversial, recent evidence strongly indicates that type 1 fimbriae, among other things, are implicated in enhancing the virulence of certain uropathogenic strains. A detailed dissection of the molecular biology of type 1 fimbriae and their role in Bacterial Adhesin would undoubtedly help to reach a clearer understanding of important aspects of Bacterial pathogenesis.