title: Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin 1 and vitronectin.
abstract: Human thrombospondin 1 (hTSP-1) is a matricellular glycoprotein facilitating bacterial adherence to and invasion into eukaryotic cells. However, the bacterial adhesin(s) remain elusive. In this study, we show a dose-dependent binding of soluble hTSP-1 to Gram-positive but not Gram-negative bacteria. Diminished binding of soluble hTSP-1 to proteolytically pretreated staphylococci suggested a proteinaceous nature of potential bacterial adhesin(s) for hTSP-1. A combination of separation of staphylococcal surface proteins by two-dimensional gel electrophoresis with a ligand overlay assay with hTSP-1 and identification of the target protein by mass spectrometry revealed the major staphylococcal autolysin Atl as a bacterial binding protein for hTSP-1. Binding experiments with heterologously expressed repeats of the AtlE amidase from Staphylococcus epidermidis suggest that the repeating sequences (R1ab-R2ab) of the N-acetyl-muramoyl-L-alanine amidase of Atl are essential for binding of hTSP-1. Atl has also been identified previously as a staphylococcal vitronectin (Vn)-binding protein. Similar to the interaction with hTSP-1, the R1ab-R2ab repeats of Atl are shown here to be crucial for the interaction of Atl with the complement inhibition and matrix protein Vn. Competition assays with hTSP-1 and Vn revealed the R1ab-R2ab repeats of AtlE as the common binding domain for both host proteins. Furthermore, Vn competes with hTSP-1 for binding to Atl repeats and vice versa. In conclusion, this study identifies the Atl repeats as bacterial adhesive structures interacting with the human glycoproteins hTSP-1 and Vn. Finally, this study provides insight into the molecular interplay between hTSP-1 and Vn, respectively, and a bacterial autolysin.
authors.affiliation: From the Department of Genetics of Microorganisms and.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Kohler
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gisch
authors.forename: Ulrike
authors.initials: U
authors.lastname: Binsker
authors.forename: Martin
authors.initials: M
authors.lastname: Schlag
authors.forename: Katrin
authors.initials: K
authors.lastname: Darm
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zähringer
authors.forename: Sven
authors.initials: S
authors.lastname: Hammerschmidt
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000201:enzymology*; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019096:Vitronectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019700:Thrombospondin 1; D019096:Vitronectin; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Adhesion; Autolysin; Extracellular Matrix Proteins; Pathogenesis; Repeats; Staphylococcus; Thrombospondin; Vitronectin