Characterization of plasmin(ogen) binding to Streptococcus pneumoniae

The proteolytic activity of plasmin promotes migration of pathogenic bacteria through the human extracellular matrix. The human pathogen Streptococcus pneumoniae binds both human plasminogen and plasmin via the surface displayed alpha-enolase designated Eno. Electron microscopic studies verified the surface exposition of the glycolytic enzyme alpha-enolase and moreover, its ability to reassociate to the cell surface. Carboxyterminal lysine residues of recently described eukaryotic and prokaryotic plasminogen-binding proteins such as SEN of S. pyogenes are involved in interaction with lysine binding sites of kringle domains of plasminogen. In this study, the role of carboxy terminal lysyl residue of eno in plasminogen binding is further analysed.

Site-directed mutagenesis of eno gene was done using DNA primers with Hind III-restriction enzyme sites for cloning. Purified Eno fusion proteins were separated by SDS-PAGE and human plasminogen binding assay was performed. Radioiodinated ligand binding was done by competitive inhibition assay.

Binding assays performed under reduced conditions indicated also a role of the C-terminal lysyl residues of Eno for plasmin(ogen) binding. Binding of pneumococci to radioiodinated plasminogen was competitively inhibited in the presence of plasminogen, kringle 1-3 (LBS 1) and the lysineanalogon epsilon-amino caproic acid indicating the crucial role of lysine-binding sites of plasminogen. However, binding analysis of plasminogen and LBS 1 to wild type Eno and carboxy terminal modified Eno proteins did not reveal any difference in plasminogen-binding activity under native conditions.

The present results suggested the presence of a further plasminogenbinding motif in Eno. This hypothesis was confirmed by plasminogen-binding activity of reassociated C-terminal modified enolase to the pneumococcal surface and indicated, therefore, the presence of a further binding motif in Eno for plasminogen binding.