Abstract Streptococcus pyogenes is a human-specific bacterial pathogen that produces several proteins that exploit the plasminogen (PLG)-plasmin (PLM) fibrinolysis system to dismantle blood clots and to facilitate spread and survival within the host. In this study, we explored the interactions between streptolysin O (SLO), a key cytolytic toxin produced by S. pyogenes , and human plasma proteins using affinity-enrichment mass spectrometry. SLO binds specifically to PLG, and this interaction accelerates the conversion of PLG to PLM by tissue-type plasminogen activator (tPA). To further investigate the molecular detail of the PLG-SLO interaction, we employed hydrogen/deuterium exchange mass spectrometry combined with targeted cross-linking mass spectrometry, uncovering that SLO binding induces local conformational shifts in PLG. These changes lead to the formation of a stabilized intermediate PLG-SLO complex that becomes significantly more sensitive to proteolytic processing by tPA. Our findings reveal a conserved moonlighting pathomechanistic role for SLO extending beyond the well characterized cytolytic activity. Additionally, the work underscores the diversity of functional proteomics in identifying and clarifying new host-pathogen interactions.

Streptolysin O Accelerates the Conversion of Plasminogen to Plasmin