Characterization of plasmin(ogen) binding to Streptococcus pneumoniae

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Pathogenic bacteria like Streptococcus pneumoniae utilize host plasminogen for extracellular matrix invasion.
• Surface-displayed alpha-enolase (Eno) on Streptococcus pneumoniae mediates binding to human plasminogen and plasmin.
• The interaction involves lysine-binding sites on plasminogen and potentially carboxy-terminal lysine residues on binding proteins.

Purpose of the Study:

• To investigate the role of the C-terminal lysyl residue of alpha-enolase (Eno) in plasminogen binding.
• To identify potential additional plasminogen-binding motifs within Eno.

Main Methods:

• Site-directed mutagenesis of the eno gene was performed.
• Purified Eno fusion proteins were analyzed using SDS-PAGE.
• Human plasminogen binding assays, including radioiodinated ligand competitive inhibition assays, were conducted.

Main Results:

• C-terminal lysyl residues of Eno contribute to plasminogen binding under reduced conditions.
• Pneumococcal binding to plasminogen is inhibited by plasminogen, kringle 1-3, and epsilon-amino caproic acid, confirming the role of lysine-binding sites.
• No difference in plasminogen-binding activity was observed between wild-type and C-terminal modified Eno under native conditions.

Conclusions:

• The results suggest the presence of an additional, previously unidentified, plasminogen-binding motif in Eno.
• This secondary binding motif is functional even when C-terminal lysines are modified, as demonstrated by binding assays with reassociated enolase.
• Eno likely possesses multiple domains for plasminogen interaction, facilitating bacterial adhesion and invasion.