Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin-1 and vitronectin [Glycobiology and Extracellular Matrices]

December 26th, 2013 by Kohler, T. P., Gisch, N., Binsker, U., Schlag, M., Darm, K., Voelker, U., Zaehringer, U., Hammerschmidt, S.

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 2D-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 bacterial binding protein for hTSP-1. Binding experiments with heterologously expressed repeats of the AtlE amidase from Staphylococcus epidermidis suggested that the repeating sequences (R1ab-R2ab) of the N-acetyl-muramoyl-L-alanine amidase (AM) of Atl are essential for binding of hTSP-1. Atl was previously also identified as a staphylococcal vitronectin (Vn)-binding protein. Similar to the interaction with hTSP-1, the (R1ab-R2ab) repeats of Atl were 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, the study provides insight into the molecular interplay between hTSP-1 and Vn, respectively, and a bacterial autolysin.
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