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Enolase of Staphylococcus lugdunensis Is a Surface-Exposed Moonlighting Protein That Binds to Extracellular Matrix
Muzaffar Hussain1, Christian Kohler2, Karsten Becker1,2,3
1Institute of Medical Microbiology, University Hospital Münster, Münster, Germany.
Abstract:
The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.
Insights
Staphylococcus lugdunensis enolase (SlEno) acts as both a metabolic enzyme and a cell surface adherence factor. This moonlighting protein promotes bacterial colonization, invasion, and virulence, suggesting it as a potential vaccine target.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Coagulase-negative staphylococci (CoNS) include *Staphylococcus lugdunensis*, a species uniquely causing severe human infections similar to *Staphylococcus aureus*.
- Bacterial colonization and invasion necessitate adherence factors, yet few *S. lugdunensis* adhesion proteins to biotic surfaces are identified.
- Understanding *S. lugdunensis* virulence mechanisms is crucial for developing effective treatments against severe infections.
Purpose of the Study:
- To investigate the dual functionality of *S. lugdunensis* enolase (SlEno) as both a metabolic enzyme and an adherence factor.
- To elucidate the role of SlEno in bacterial adhesion to extracellular matrix proteins and its contribution to virulence.
- To assess the potential of SlEno as a therapeutic target for preventing *S. lugdunensis* infections.
Main Methods:
- Phylogenetic analysis of SlEno to determine its evolutionary relationship with other staphylococcal enolases.
- Matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot to confirm SlEno localization (cytoplasm and cell surface).
- In vitro assays to assess SlEno's binding to extracellular matrix proteins (laminin, fibronectin, fibrinogen, collagen IV) and plasminogen, and its role in bacterial migration and host immune cell clearance.
Main Results:
- SlEno exhibits conserved enolase activity and functions as an adherence factor, binding strongly to laminin, fibronectin, fibrinogen, and collagen IV.
- SlEno binds plasminogen and enhances its activation by tissue plasminogen activator, facilitating bacterial migration through fibrin matrices.
- Surface-exposed SlEno enhances bacterial clearance by granulocytes when antibodies against SlEno are present.
Conclusions:
- *S. lugdunensis* enolase possesses a moonlighting function, acting as a significant virulence factor for tissue colonization and invasion.
- SlEno's role in adhesion, extracellular matrix interaction, and immune evasion highlights its importance in *S. lugdunensis* pathogenesis.
- SlEno represents a promising vaccine candidate for preventing severe infections caused by *S. lugdunensis*.
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