The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) γ-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin αIIbβ3 and ClfA bind to the same segment in the Fg γ-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the “Dock, Lock and Latch” mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not αIIbβ3 may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.
Uncontrolled hemorrhage accounts for more than 30% of trauma deaths worldwide. Current hemostatic devices focus primarily on time to hemostasis, but prevention of bacterial infection is also critical for improving survival rates. In this study, we sought to improve on current devices used for hemorrhage control by combining the large volume-filling capabilities and rapid clotting of shape memory polymer (SMP) foams with the swelling capacity of hydrogels. In addition, a hydrogel composition was selected that readily complexes with elemental iodine to impart bactericidal properties to the device. The focus of this work was to verify that the advantages of each respective material (SMP foam and hydrogel) are retained when combined in a composite device. The iodine-doped hydrogel demonstrated an 80% reduction in bacteria viability when cultured with a high bioburden of Staphylococcus aureus. Hydrogel coating of the SMP foam increased fluid uptake by 19X over the uncoated SMP foam. The composite device retained the shape memory behavior of the foam with more than 15X volume expansion after being submerged in 37°C water for 15 minutes. Finally, the expansion force of the composite was tested to assess potential tissue damage within the wound during device expansion. Expansion forces did not exceed 0.6 N, making tissue damage during device expansion unlikely, even when the expanded device diameter is substantially larger than the target wound site. Overall, the enhanced fluid uptake and bactericidal properties of the shape memory foam composite indicate its strong potential as a hemostatic agent to treat non-compressible wounds.
Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, ␣11, ␣21, ␣101, and ␣111. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2 GLPGER and Scl2 GFPGER bound to recombinant human ␣1 and ␣2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrinbinding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2 GFPGEN shows specificity toward the ␣1 I-domain and does not bind the ␣2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target ␣11 and ␣21.Collagen is a major constituent of the extracellular matrix where it functions as a structural component. In addition, collagen can directly or indirectly interact with cellular receptors and regulate a variety of cellular processes (1). There are at least 28 identified mammalian collagens each consisting of three polypeptides that can be genetically identical or distinct (2). A defining feature of collagens is the tightly packed left-handed triple helix made of polypeptide segments with repeating GXY triplets. The small Gly residue fits in the interior of the triple helix, and the X and Y positions are often occupied by proline and hydroxyproline residues. Hydroxylation of proline residues stabilizes the triple helical structure and inhibition of posttranslational hydroxylation decreases the melting temperature of the mammalian collagen triple helix by ϳ15°C (3).Surface proteins with collagen-like domains recently have been found on a number of prokaryotic organisms, including Streptococcus pyogenes, Streptococcus equi, and Bacillus anthracis (4 -6). These collagen-like domains contain conserved GXY repeats but lack the hydroxyproline found in mammalian collagens. S. pyogenes often contain two collagen-like proteins, Scl1 and Scl2 (4, 7-13). The primary sequences and length of different domains, including the GXY repeat segments, vary considerably in Scl1 and Scl2 proteins from different strains (4). In electron micrographs, both proteins form a lollipop-like structure with an N-terminal variable globular domain connected to an extende...
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