A Novel Virulence Mechanism in Staphylococcus aureus: FnBPA Cross-Linking to Fibrin(ogen) and Fibronectin by Bacterially Activated FXIII and TG2

Staphylococcus aureus is a versatile opportunistic pathogen capable of causing a spectrum of infections, from superficial skin lesions to life-threatening systemic diseases. Its capacity to evade host immune responses and establish persistent infections is critically dependent on its ability to adhere to and colonize host tissues. Central to this process is the interaction of S. aureus surface proteins with extracellular matrix (ECM) components, particularly fibronectin (Fn) and fibrinogen (Fbg). This thesis investigates a novel pathogenic mechanism by which S. aureus exploits host transglutaminases, specifically Factor XIII (FXIII) and tissue transglutaminase 2 (TG2), to covalently anchor itself to host proteins through the multifunctional surface adhesin Fibronectin Binding Protein A (FnBPA), with the bacterial coagulase von Willebrand factor-binding protein (vWbp) playing a central activating role. The study begins by examining the expression profile and localization of vWbp during S. aureus growth, revealing that the protein is secreted during the exponential phase and subsequently rebinds to the bacterial surface via interactions with lipoteichoic acid (LTA) and peptidoglycan (PPG). Functionally, surface-associated vWbp non-proteolytically activates prothrombin, leading to fibrin formation and subsequent activation of FXIII. In this environment, bacterially-activated FXIII catalyzes the formation of ε-(γ-glutamyl)-lysine isopeptide bonds between Fbg and FnBPA, embedding the bacterium within a stable fibrin scaffold. Biochemical analysis identified Gln103 in the N1 subdomain of FnBPA's N-terminal Region A as the key reactive glutamine residue essential for vWbp-activated FXIII mediated crosslinking. Recombinant FnBPA mutants lacking Gln103 (Q103A) retained non-covalent binding to Fbg via the classical Dock, Lock, and Latch (DLL) mechanism but failed to form covalent complexes with fibrin(ogen). This specificity was confirmed using whole bacterial cells expressing Q103A FnBPA, which demonstrated impaired incorporation into fibrin matrices. Also, bacterial virulence was significantly reduced in a murine subcutaneous infection model in presence of the Q103A mutation, as evidenced by smaller dermonecrotic lesions and lower bacterial loads compared to wild-type strains. In addition, this study also establishes that TG2, a transglutaminase expressed in various tissues and induced during inflammation, can catalyze the same crosslinking reaction, broadening the physiological relevance of this mechanism beyond sites of coagulation. Beyond Fbg, this work demonstrates that Fn is also a substrate for FnBPA-mediated covalent crosslinking to host proteins in the presence of vWbp-activated FXIII. Interestingly, this function is conferred not by the N1N2N3 region, but by the C-terminal repeat region (RR) of FnBPA, which forms high-molecular-weight complexes with both full-length Fn and the N-terminal 29 kDa fragment of Fn. The RR region is already known to mediate non-covalent interactions with Fn through a tandem β-zipper mechanism, highlighting its dual role in adhesive processes. In summary, this thesis uncovers a novel virulence mechanism whereby S. aureus utilizes vWbp to hijack host transglutaminase activity, resulting in covalent stabilization of bacterial-host protein complexes via FnBPA. These findings not only deepen our understanding of S. aureus adhesion biology and immune evasion but also identify FnBPA-mediated covalent crosslinking as a promising therapeutic target, particularly in the context of antibiotic-resistant infections and biofilm-associated diseases.