Il ruolo dei recettori del plasminogeno FnBPA e FnBPB di Staphylococcus aureus nella promozione della trasmigrazione batterica attraverso coaguli di fibrina

Staphylococcus aureus, a Gram-positive pathogenic bacterium, represents the etiologic agent of potentially harmful human health diseases such as sepsis, endocarditis and necrotizing pneumonia, and is also responsible for many less severe clinical manifestations such as epithelial and mucosal infections. Nowadays, the emergence of methicillin- and vancomycin-resistant S. aureus strains, is of great concern. Despite the advance in antibiotic development, treating these infections remains a huge challenge for the scientific community. The main class of staphylococcal proteins responsible for host tissue colonization is represented by the cell wall-anchored (CWA) proteins. Among them, the major family is the MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Like several other invasive pathogens, S. aureus can capture human plasma protein plasminogen, a 92 kDa zymogen, and trigger its conversion to catalytically active plasmin mediated by host plasminogen activators, tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA) or by staphylococcal plasminogen activator, staphylokinase (SAK). Plasmin controls several processes such as fibrinolysis, wound healing and tissue remodelling as well as degradation of the complement opsonin C3b and the immunoglobulin Fc domain. Thus, binding of PLG to the surface of S. aureus and its activation to plasmin renders bacteria into proteolytic organisms capable of degrading extracellular matrix (ECM) and other key components of host defence machinery. In this study, we have shown that S. aureus LAC srtA, a mutant defective in the sortase enzyme, which is responsible for anchoring CWA proteins to the S. aureus cell surface, captured 10-fold less PLG compared to the wild type LAC strain, suggesting that one or more CWA proteins is involved. Among several recombinant CWA proteins of S. aureus screened for PLG binding, two CWA proteins, the fibrinogen and fibronectin binding proteins A and B, were identified as potential “PLG receptors”. The molecular aspects of the interaction of these two staphylococcal proteins with PLG were elucidated. Analysis of proteins-ligand binding indicates a high-affinity interaction in the nanomolar range (KD = 0,532 μM). Kringle 4 of PLG was predicted to be the binding domain within the zymogen for fibronectin-binding proteins. Analysis of PLG capture by S. aureus LAC ΔfnbA/B, a mutant that lacks both FnBPA and FnBPB, revealed that fibronectin-binding proteins are the main PLG receptors expressed on the surface of the bacterium, even though other surface proteins are involved in PLG binding. PLG captured on the surface of S. aureus or Lactococcus lactis expressing heterologous FnBPB could be activated by exogenous (tPA) or endogenous (SAK) plasminogen activators to the broad spectrum proteolytic enzyme plasmin. Plasmin bound to the surface of the bacterium was functionally active and cleaved the artificial substrate S2251 and human fibrinogen (FBG), indicating that the presence of active plasmin on the surface of S. aureus cells most likely contributes to the pathogenesis. To further investigate this aspect, we used an in vitro system aimed to elucidate the fibrinolytic activity displayed by the pathogen in the human host. Indeed, the Transwell® system apparatus is a vertical side by side diffusion system across a semipermeable microporous membrane, which allows for free passage of diffusible factors between two compartments (lumen and albumen). Fibrin clots were assembled on the surface of the microporous membrane in order to test bacterial transmigration through the clots. Taking advantage of this system, we showed that plasmin derived from the activation of PLG captured by FnBPs on the surface of S. aureus cleaves fibrin in blood clots, ensuring bacterial transmigration.