Staphyloccoccus aureus is a Gram-positive pathogenic bacterium representing a major human health threat worldwide. S. aureus infection can lead to many different clinical manifestations, from the less severe epithelial and mucosal infections to the most harmful, sometimes lethal sepsis, endocarditis, necrotizing fasciitis or toxic shock syndrome; it can also provoke clinical complications on patients affected by other diseases, such as cystic fibrosis. Also, the rising concern about staphylococcal infections resides on its remarkable ability to acquire antibiotic resistance, mostly through horizontal gene transfer, chromosomal mutations or antibiotic selection. Particularly, the main concern with S. aureus is the extent of the emergence of methicillin- (MRSA) and vancomycin-resistant (VRSA) strains. S. aureus can colonize host tissues by expressing a wide variety of surface proteins called cell wall-anchored (CWA) proteins. The most relevant class of CWAs is the MSCRAMMs family (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Human immune system can count on several antimicrobial mechanisms, a significant example of which is represented by NETs (neutrophil extracellular traps). Among these, histones play a major role for the neutralization of endotoxins and destruction of cell membrane, eventually leading to the eradication of bacterial infections, including the staphylococcal one. Nevertheless, this highly adaptable bacterium has developed a defencse from histones-mediate killing: S. aureus surface proteins fibronectin-binding proteins A and B (FnBPbpA/B) can capture Plasminogen (PLG), a zymogen protein produced by the liver and released in the blood system. PLG can be converted into the serine protease Plasmin (Plm) by tissue plasminogen activator (tPA) and by urokinase plasminogen activator (uPA) or by the staphylococcal-expressed enzyme Staphylokinase (SAK). Plasmin performs fibrinolytic and proteolytic functions by which it can, respectively, break down fibrin clots and degrades immunoglobulin G (IgG), complement system element C3b and, as we have demonstrated, also all classes of calf thymus histones (CTH). Therefore, plasmin-bound S. aureus can survive extracellular histones-mediate killing thanks to the proteolytic activity of the enzyme. In this study, we evaluated particularly how staphylococcal survival to histones can increase once S. aureus interacts with plasminogen. We performed a number of comparative experiments between Bacterial cultures, wild-type strain and mutant strains deficient in some key genes (∆sak and ∆srtA), incubated or not with PLG and/or its singular domains (Kringles), in presence or absence of PLG activators, were evaluated for bacterial survival to histones. Biochemical analysis on histones themselves were then performed, observing in which specific conditions PLG can be efficiently activated or not, and whether histones degradation can be performed totally or partially by active plasmin. Histones resulted to have an efficient bactericidal activity unless in presence of PLG combined with activators. Comparison between wild-type strain and its mutants ∆sak and ∆srtA, lead to different outcomes, but not always significant. Finally, we tested which might be the surface proteins majorly involved in plasminogen binding and, about the latter, which molecular domains might be involved for histones interaction and eventually degradation. Our results confirm the assumptions of the many previous studies about S. aureus survival to histones and so its potential resistance to human’s NETs, defining its abilities to escape immune defences and its role as a major infectious treat for human beings. Finally, we found potentially useful details for further studies about the mechanism of bacterium-PLG-activator interaction and the derived advantage that the microorganism obtains.
Lo Staphylococcus aureus è un batterio Gram-positivo e rappresenta una grave minaccia globale. L'infezione da S. aureus può portare adiverse manifestazioni cliniche, dalle infezioni epiteliali e mucose meno gravi alle più nocive, a volte letali, sepsi endocarditi, fascite necrotizzante o alla sindrome da shock tossico; può anche provocare complicanze cliniche su pazienti affettida altre malattie, come la fibrosi cistica. Inoltre, la crescente preoccupazione perle infezioni da stafilococchi risiede nella sua notevole capacità di acquisire resistenza agli antibiotici, soprattutto attraverso il trasferimento genico orizzontale, le mutazioni cromosomiche o la selezione di antibiotici. La principale preoccupazione per S. aureus è l'entità dell'emergenza di ceppi resistenti alla meticillina (MRSA) e alla vancomicina (VRSA). S. aureus può colonizzare i tessuti dell'ospite esprimendo un'ampia varietà di proteine di superficie chiamate CWA (cell wall-anchored). La classe più rilevante di CWA è la famiglia MSCRAMM (Microbial SurfaceComponents Recognizing AdhesiveMatrix Molecules). Il sistema immunitario umano può contare su diversi meccanismi antimicrobici, un esempio significativo di questi sono le NET (trappole extracellulari di neutrofili). Tra i componenti delle NET, gli istoni svolgono un ruolo importante per la neutralizzazione di endotossine e la distruzione della parete cellulare, portando all'eradicazione delle infezioni batteriche, compresa quella stafilococcica. Tuttavia, questo batterio altamente adattabile ha sviluppato una difesa contro gli istoni: S.aureus possiede proteine di superficie,fibronectin-binding protein A e B(FnBPbpA/B), in grado di catturare Plasminogeno (PLG),uno zimogeno prodotto dal fegato e rilasciato nel sistema sanguigno. Il PLG può essere convertito nella proteasi serinica Plasmina (Plm) dall'attivatore plasminogeno tessutale (tPA) e dall'attivatore plasminogeno dell'urochinasi (uPA) o dall'enzima stafilococcico Staphylokinase(SAK). La plasmina svolge funzioni fibrinolitiche e proteolitiche attraverso le quali può scindere i coaguli di fibrina, degradare l'immunoglobulina G (IgG), l' elemento di sistema di complemento C3b e, come abbiamo dimostrato, tutte le classi di istoni del timo del vitello(CTH). Pertanto, il S. aureus legato alla plasmina può sopravvivere agli istoni extracellulari, uccidendoli grazie all'attività proteolitica dell'enzima. In questo studio, abbiamo analizzato come la sopravvivenza stafilococcica agli istoni possa aumentare una volta che S. aureus interagisce con il plasminogeno. Abbiamo condotto una serie di esperimenti comparativi tra colture batteriche, ceppi wild-type e ceppi mutanti carenti di alcuni geni chiave(∆sak e ∆srtA), incubati o meno conPLG e/o i suoi domini singoli(Kringles), in presenza o assenza di attivatori PLG, valutando la sopravvivenza batterica agli istoni. Sono state poi effettuate analisi biochimiche sugli istoni stessi, osservando in quali condizioni PLG può essere efficacemente attivato o meno, e se la degradazione degli istoni può essere eseguita totalmente o meno da plasmina. Gli istoni hanno mostrato un'attività battericida efficiente a meno che in presenza di PLG combinato con attivatori. Il confronto tra il ceppo wild-type e i suoi mutanti ∆sak e ∆srtA, porta a risultati diversi, ma non sempre significativi. Infine, abbiamo testato quali potrebbero essere le proteine di superficie maggiormente coinvolte nel legame con plasminogeno e, su quest'ultimo, quali domini molecolari potrebbero essere coinvolti nell’interazione e degradazione di istoni. I nostri risultati confermano i presupposti dei molti studi precedenti sulla sopravvivenza di S. aureus agli istoni e quindi la sua resistenza alle NET, definendo le sue capacità di evadere le difese immunitarie e il suo ruolo di importante patogeno. Infine, abbiamo trovato dettagli utili per ulteriori studi sul meccanismo di interazione batterio-PLG-attivatore.
Staphylococcus aureus sfugge all'azione battericida degli istoni tramite plasminogeno attivo
MAIOLATESI, DIEGO
2017/2018
Abstract
Staphyloccoccus aureus is a Gram-positive pathogenic bacterium representing a major human health threat worldwide. S. aureus infection can lead to many different clinical manifestations, from the less severe epithelial and mucosal infections to the most harmful, sometimes lethal sepsis, endocarditis, necrotizing fasciitis or toxic shock syndrome; it can also provoke clinical complications on patients affected by other diseases, such as cystic fibrosis. Also, the rising concern about staphylococcal infections resides on its remarkable ability to acquire antibiotic resistance, mostly through horizontal gene transfer, chromosomal mutations or antibiotic selection. Particularly, the main concern with S. aureus is the extent of the emergence of methicillin- (MRSA) and vancomycin-resistant (VRSA) strains. S. aureus can colonize host tissues by expressing a wide variety of surface proteins called cell wall-anchored (CWA) proteins. The most relevant class of CWAs is the MSCRAMMs family (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Human immune system can count on several antimicrobial mechanisms, a significant example of which is represented by NETs (neutrophil extracellular traps). Among these, histones play a major role for the neutralization of endotoxins and destruction of cell membrane, eventually leading to the eradication of bacterial infections, including the staphylococcal one. Nevertheless, this highly adaptable bacterium has developed a defencse from histones-mediate killing: S. aureus surface proteins fibronectin-binding proteins A and B (FnBPbpA/B) can capture Plasminogen (PLG), a zymogen protein produced by the liver and released in the blood system. PLG can be converted into the serine protease Plasmin (Plm) by tissue plasminogen activator (tPA) and by urokinase plasminogen activator (uPA) or by the staphylococcal-expressed enzyme Staphylokinase (SAK). Plasmin performs fibrinolytic and proteolytic functions by which it can, respectively, break down fibrin clots and degrades immunoglobulin G (IgG), complement system element C3b and, as we have demonstrated, also all classes of calf thymus histones (CTH). Therefore, plasmin-bound S. aureus can survive extracellular histones-mediate killing thanks to the proteolytic activity of the enzyme. In this study, we evaluated particularly how staphylococcal survival to histones can increase once S. aureus interacts with plasminogen. We performed a number of comparative experiments between Bacterial cultures, wild-type strain and mutant strains deficient in some key genes (∆sak and ∆srtA), incubated or not with PLG and/or its singular domains (Kringles), in presence or absence of PLG activators, were evaluated for bacterial survival to histones. Biochemical analysis on histones themselves were then performed, observing in which specific conditions PLG can be efficiently activated or not, and whether histones degradation can be performed totally or partially by active plasmin. Histones resulted to have an efficient bactericidal activity unless in presence of PLG combined with activators. Comparison between wild-type strain and its mutants ∆sak and ∆srtA, lead to different outcomes, but not always significant. Finally, we tested which might be the surface proteins majorly involved in plasminogen binding and, about the latter, which molecular domains might be involved for histones interaction and eventually degradation. Our results confirm the assumptions of the many previous studies about S. aureus survival to histones and so its potential resistance to human’s NETs, defining its abilities to escape immune defences and its role as a major infectious treat for human beings. Finally, we found potentially useful details for further studies about the mechanism of bacterium-PLG-activator interaction and the derived advantage that the microorganism obtains.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.
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https://hdl.handle.net/20.500.14239/24985