Staphylococcus aureus is an important opportunistic human pathogen that persistently colonizes about 20% of the human population and can cause a variety of diseases including skin infections, pneumonia, endocarditis, and sepsis. The emergence of methicillin- and vancomycin resistance among clinical isolates of S. aureus has made treatment of staphylococcal infections difficult, reviving research for a vaccine to prevent infections especially in patients who are at high risk. In the last few years several genomic and proteomic studies of S. aureus have provided an amounts of possible targets for vaccine design. Among them, significant importance is given to S. aureus surface proteins known as Cell Wall Anchored (CWA) proteins, or adhesins that are covalently anchored to the cell wall peptidoglycan. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. The main family that represents them is the one of MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Among the multitude of interactions with host proteins, S. aureus binds the human plasma protein plasminogen (PLG), a 92-kDa zymogen, which can be cleaved and activated to plasmin by tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Along with PLG receptors S. aureus co-expresses a PLG activator, staphylokinase (SAK) that changes the conformation of PLG and facilitates its activation by surrounding plasmin molecules. Plasmin controls several processes such as fibrinolysis, wound healing and tissue remodelling. Thus, binding of PLG to the surface of S. aureus and its activation to plasmin turns bacteria into proteolytic organisms capable of degrading extracellular matrix (ECM) andbasement membranes. Furthermore plasmin can cleave native C3 leading to the formation of the C3b, which is subsequently degraded and inactivated to iC3b. In this study several recombinant CWA proteins were screened for binding to PLG. We focused our attention on the binding of PLG to FnBPB, a fibronectin/fibrinogen binding protein, providing evidence on the molecular aspects of this interaction in comparison with fibrinogen. The binding appeared specific since no interaction was observed with other CWA proteins such as ClfA/B, IsdB, Bbp and SdrD. Analysis of PLG binding to FnBPB indicates a high affinity interaction in the nanomolar range of KD. The mechanism through which FnBPB binds PLG is different to the one employed in the Fbg binding and is not based on the “Dock, lock and latch” model. The combination of the subdomains N2-N3 of FnBPB represents the minimal segment of region A with binding ability to recognize and bind fibrinogen and PLG. When the single subdomains N2 and N3 were tested for fibrinogen binding, none of these subdomains bound to fibrinogen. On the contrary, the subdomain N3 but not the the N2 subdomain still kept intact the ability to interact with PLG, suggesting that the molecular requirements needed for FnBPB/PLG interactions substantially differ from those involving fibrinogen binding. From literature we know that PLG binding to its natural ligands is attributed to strong affinity of Kringle domain(s) for lysine rich regions of target proteins. We investigated the role of FnBPB lysine residues in the PLG binding, finding that variants of the recombinant A domain with substitutions at two distinct conserved lysine-rich sites in subdomain N3 bound PLG with reduced affinity. We also demonstrated that FnBPB-bound PLG is accessible to human tPA (tissue plasminogen activator), uPA and bacterial SAK (staphylokinase) and can be activated to plasmin.
Staphylococcus aureus, un importante patogeno umano che colonizza circa il 20% della popolazione , può causare diverse patologie quali infezioni della pelle, polmonite, endocarditi e sepsi. La comparsa e diffusione ceppi meticillina e vancomicina resistenti individuati in diversi isolati clinici rendono sempre più difficile il trattamento di infezioni da stafilococco. Questo ha portato ad un rinnovato interesse nella ricerca di vaccini per prevenire tali infezioni. Negli ultimi anni molti possibili target sono stati individuati grazie a studi di genomica e proteomica riguardanti S. aureus. Tra i possibili target una rilevanza particolare è stata data alle proteine di superficie di S. aureus, conosciute come Cell Wall Anchored (CWA) proteins, o adesine, covalentemente ancorate ai peptidoglicani della parete cellulare batterica. Queste adesine svolgono numerose funzioni, incluse l’adesione a cellule o tessuti dell’ospite, evasione della risposta immunitaria e formazione di biofilm. Da ciò si deduce che le proteine batteriche di superficie sono fattori di virulenza essenziali per la sopravvivenza di S. aureus sia nello stato commensale, sia durante l’infezione, e che utilizzarle come target per vaccini futuri può aiutare a combattere le infezioni da stafilococco. MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) rappresenta la famiglia principale delle adesine. S. aureus interagisce con una grande varietà di proteine dell’ospite, tra cui il plasminogeno (PLG), uno zimogeno che può essere attivato a plasmina grazie all’azione dell’attivatore tissutale del plasminogeno (tPA), o dell’urochinasi (uPA). S. aureus, oltre ai recettori per il plasminogeno, esprime anche un attivatore del plasminogeno, la stafilochinasi (SAK), che attraverso un cambiamento conformazionale del plasminogeno ne favorisce l’attivazione a plasmina. La plasmina controlla diversi processi quali: la fibrinolisi, guarigione delle ferite e rimodellamento tissutale. Perciò il legame del PLG alla superficie di S. aureus e la sua successiva attivazione a plasmina, rende i batteri organismi proteolitici, capaci di degradare la matrice extracellulare (ECM) e le membrane basali. La plasmina inoltre può formare, attraverso taglio proteolitico del C3, il C3b successivamente degradato e inattivato a iC3b. In questo lavoro di tesi è stato fatto uno screen di diverse proteine CWA ricombinanti per individuare possibili adesine leganti il PLG. La nostra attenzione si è poi focalizzata su FnBPB (fibronectin/fibrinogen binding protein), della quale abbiamo mostrato gli aspetti molecolari dell’interazione con il PLG rispetto al legame con il fibrinogeno (Fbg). Il legame si è dimostrato specifico, in quanto altre proteine CWA considerate non mostravano interazioni con il PLG. Successive analisi hanno evidenziato un’alta affinità nel legame tra FnBPB e PLG ed è stato appurato che tale interazione avviene con un meccanismo diverso rispetto al modello “dock, lock and latch” valido per il legame tra FnBPB e Fbg. È stato inoltre osservato che la combinazione dei sottodomini N2-N3 di FnBPB rappresenta il segmento minimo della regione A con capacità di riconoscere e legare non solo il fibrinogeno ma anche il plasminogeno e che dei singoli sottodomini N2 e N3, nessuno era in grado di legare il Fbg, se testato singolarmente, mentre il singolo sottodominio N3 risultava coinvolto nel legame al PLG. Dalla letteratura è noto che il legame tra il plasminogeno e i suoi substrati naturali è dovuto alla forte affinità dei domini Kringle del PLG per regioni ricche di lisine presenti sulle proteine target. Abbiamo quindi analizzato il ruolo di alcune lisine esposte di FnBPB nel legame con il PLG, dimostrando che varianti ricombinanti del dominio A con sostituzioni in due distinte regioni del sottodominio N3 ricche in lisine, legano il plasminogeno con una ridotta affinità.
Identificazione e caratterizzazione di un sito di legame al plasminogeno in FnBPB, un'adesina di Staphylococcus aureus che lega fibrinogeno/fibronectina
GALLOTTA, LUCREZIA
2015/2016
Abstract
Staphylococcus aureus is an important opportunistic human pathogen that persistently colonizes about 20% of the human population and can cause a variety of diseases including skin infections, pneumonia, endocarditis, and sepsis. The emergence of methicillin- and vancomycin resistance among clinical isolates of S. aureus has made treatment of staphylococcal infections difficult, reviving research for a vaccine to prevent infections especially in patients who are at high risk. In the last few years several genomic and proteomic studies of S. aureus have provided an amounts of possible targets for vaccine design. Among them, significant importance is given to S. aureus surface proteins known as Cell Wall Anchored (CWA) proteins, or adhesins that are covalently anchored to the cell wall peptidoglycan. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. The main family that represents them is the one of MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Among the multitude of interactions with host proteins, S. aureus binds the human plasma protein plasminogen (PLG), a 92-kDa zymogen, which can be cleaved and activated to plasmin by tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Along with PLG receptors S. aureus co-expresses a PLG activator, staphylokinase (SAK) that changes the conformation of PLG and facilitates its activation by surrounding plasmin molecules. Plasmin controls several processes such as fibrinolysis, wound healing and tissue remodelling. Thus, binding of PLG to the surface of S. aureus and its activation to plasmin turns bacteria into proteolytic organisms capable of degrading extracellular matrix (ECM) andbasement membranes. Furthermore plasmin can cleave native C3 leading to the formation of the C3b, which is subsequently degraded and inactivated to iC3b. In this study several recombinant CWA proteins were screened for binding to PLG. We focused our attention on the binding of PLG to FnBPB, a fibronectin/fibrinogen binding protein, providing evidence on the molecular aspects of this interaction in comparison with fibrinogen. The binding appeared specific since no interaction was observed with other CWA proteins such as ClfA/B, IsdB, Bbp and SdrD. Analysis of PLG binding to FnBPB indicates a high affinity interaction in the nanomolar range of KD. The mechanism through which FnBPB binds PLG is different to the one employed in the Fbg binding and is not based on the “Dock, lock and latch” model. The combination of the subdomains N2-N3 of FnBPB represents the minimal segment of region A with binding ability to recognize and bind fibrinogen and PLG. When the single subdomains N2 and N3 were tested for fibrinogen binding, none of these subdomains bound to fibrinogen. On the contrary, the subdomain N3 but not the the N2 subdomain still kept intact the ability to interact with PLG, suggesting that the molecular requirements needed for FnBPB/PLG interactions substantially differ from those involving fibrinogen binding. From literature we know that PLG binding to its natural ligands is attributed to strong affinity of Kringle domain(s) for lysine rich regions of target proteins. We investigated the role of FnBPB lysine residues in the PLG binding, finding that variants of the recombinant A domain with substitutions at two distinct conserved lysine-rich sites in subdomain N3 bound PLG with reduced affinity. We also demonstrated that FnBPB-bound PLG is accessible to human tPA (tissue plasminogen activator), uPA and bacterial SAK (staphylokinase) and can be activated to plasmin.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/19509