Streptococcus agalactiae, also known as group B Streptococcus (GBS), corresponds to a species of spherical Gram-positive bacteria often organized in pairs or chains. Initially, it was thought that pathogenic infections of S. agalactiae were just present in mammalian species. However, nowadays there are many diseases linked to this pathogen in fishes, reptiles and frogs. Also, S. agalactiae is usually found in the free environment, such as in milk and dairy herds’ feces. Although this species is frequently associated with the normal nonvirulent human microbiota, it can also be related to severe diseases in its invasive form. The cell wall of pathogenic bacteria is normally associated with many virulence factors, and, besides the capsule, flagella and pilus, Gram-positive bacteria also have many surface proteins responsible for recognizing and binding to eukaryotic molecules, such as adhesins, toxins, proteases, and invasins. Two bacterial adhesins with notable influence in S. agalactiae virulence are the serine rich repeat glycoproteins Srr1 and Srr2. These proteins are known to interact with human fibrinogen and plasminogen. Importantly, Srr2 has a higher affinity to fibrinogen compared with Srr1 and is only associated with S. agalactiae ST-17 hypervirulent isolates. Considering this, the main focuses of this work were referred to the characterization of the interaction between Srr2 and human fibronectin, a big glycoprotein present in the extracellular matrix and in the blood plasma, important for wound healing, cell adhesion and migration. The experiments chosen to characterize the interaction between Srr2 and fibronectin were based on the use of biochemical techniques, such as ELISA, western blot, dot blot, affinity chromatography, BCA protein assay, and SDS-PAGE. Through a comparison between ST-17 and non-ST-17 S. agalactiae strains, it was identified that Srr2 promotes specific binding of ST-17 strains to fibronectin. By using a recombinant Srr2 binding region (rSrr2-BR) produced through affinity chromatography, it was possible to evaluate that the binding affinity between rSrr2-BR and fibronectin differs when using immobilized rSrr2-BR or immobilized fibronectin. In this sense, it is feasible to hypothesize that some specific conformational changes of one or both proteins can be involved in their binding affinity. Furthermore, through western blot and dot blot analysis, it was shown that the binding site of rSrr2-BR on fibronectin is probably represented by its cell-binding domain (CBD) fragment. Moreover, ELISA assays conducted applying different concentrations of NaCl revealed that ionic forces play an important role in the interaction of Srr2 with fibronectin, as already shown to occur with the binding of Srr2 with fibrinogen. Finally, the evaluation of the interaction of mutant forms of rSrr2-BR with fibronectin allowed the identification of the dock, lock and latch (DLL) binding mechanism for this interaction, as previously characterized for Srr2 binding to fibrinogen as well. In conclusion, this study promotes the characterization of a new important interaction between S. agalactiae, an emerging pathogen, and human fibronectin, revealing valuable information surrounding this topic. Studies conducted investigating protein-protein interactions are extremely important for the future development of new drugs and vaccines. Knowing this, through the analysis of the Srr2 structure and its interaction with fibronectin, this virulence factor can be considered a promising candidate for drug targeting as it is exclusively associated with hypervirulent strains resistant to many antimicrobial agents. These S. agalactiae strains represent a huge economic and health care system problem nowadays. So, by focusing research on these topics, we can mitigate the spread of S. agalactiae and the future consequences related to this issue.
Streptococcus agalactiae, noto anche come Streptococco di gruppo B (GBS), corrisponde a una specie di batteri Gram-positivi spesso organizzati in coppie o catene. S. agalactiae si trova di solito nell'ambiente libero, ad esempio nel latte e nelle feci bovine. Sebbene si tratti di un commensale del tratto gastro-intestinale e uro-genitale degli adulti sani, questa specie può anche diventare patogena ed essere causa di malattie gravi. La parete cellulare dei batteri patogeni dispone normalmente di molti fattori di virulenza. Oltre alla capsula, ai flagelli e ai pili, i batteri Gram-positivi hanno anche molte proteine di superficie responsabili del riconoscimento e del legame con diverse molecole eucariotiche, ad esempio adesine, tossine, proteasi e invasine, importanti per l'invasione e la patogenicità dei batteri Gram-positivi. Due adesine batteriche con una notevole influenza sulla virulenza di S. agalactiae sono le glicoproteine con ripetizioni ricche di serine Srr1 e Srr2. Queste proteine sono note per interagire con il fibrinogeno ed il plasminogeno umano. Srr2 ha un'affinità maggiore per il fibrinogeno rispetto a Srr1 ed è associata solo agli isolati ipervirulenti ST-17 di S. agalactiae. In considerazione di ciò, l'obiettivo principale di questo lavoro è stato la caratterizzazione dell'interazione tra Srr2 e la fibronectina umana, una glicoproteina presente nella matrice extracellulare e nel plasma sanguigno, importante nei processi di cicatrizzazione delle ferite. Gli esperimenti scelti per studiare l’interazione tra la fibronectina e Srr2 si sono basati sull'uso di tecniche biochimiche, come ELISA, western blot, dot blot, cromatografia di affinità, saggio proteico BCA e SDS-PAGE. Attraverso un confronto tra ceppi ST-17 e non ST-17 di S. agalactiae, è stato identificato che Srr2 promuove il legame specifico dei ceppi ST-17 alla fibronectina. Inoltre, utilizzando una regione di legame ricombinante di Srr2 (rSrr2-BR) purificata tramite cromatografia di affinità, è stato possibile valutare che l'affinità di legame tra rSrr2-BR e fibronectina differisce quando si utilizza rSrr2-BR immobilizzato o fibronectina immobilizzata. In questo senso, è possibile ipotizzare che alcuni specifici cambiamenti conformazionali di una o entrambe le proteine possano essere coinvolti nella loro affinità di legame. Inoltre, attraverso analisi di western blot e dot blot, è stato dimostrato che il sito di legame di rSrr2-BR sulla fibronectina è rappresentato dal dominio di legame cellulare (CBD) di quest’ultima. Inoltre, saggi ELISA condotti applicando concentrazioni crescenti di NaCl hanno rivelato che le forze ioniche giocano un ruolo importante nell'interazione di Srr2 con la fibronectina, come già dimostrato per il legame di Srr2 con il fibrinogeno. Infine, la valutazione dell’interazione di forme mutanti di rSrr2-BR con la fibronectina ha permesso di individuare il modello di legame dock, lock and latch (DLL) per tale interazione, già precedentemente caratterizzato per il legame di Srr2 con il fibrinogeno. In conclusione, questo studio ha promosso una nuova visione delle interazioni di S. agalactiae, un patogeno emergente, con la fibronectina umana, rivelando informazioni preziose su questa interazione. Gli studi sulle interazioni proteina-proteina sono estremamente importanti per lo sviluppo futuro di nuovi farmaci e vaccini. In considerazione di ciò, attraverso l'analisi della struttura di Srr2 e della sua interazione con la fibronectina, questo fattore di virulenza può essere considerato un promettente candidato per lo sviluppo di nuove terapie, poiché sarebbero dirette contro ceppi ipervirulenti, negli ultimi tempi sempre più resistenti a diversi agenti antimicrobici. Pertanto, concentrare la ricerca su questi argomenti, potrebbe contribuire a mitigare la diffusione di S. agalactiae e le conseguenze future legate a questo problema.
Caratterizzazione dell'interazione della glicoproteina con ripetizioni ricche di serine (Srr2) di Streptococcus agalactiae con la fibronectina dell'ospite
BELLAN MENEGUSSI, ELISA
2023/2024
Abstract
Streptococcus agalactiae, also known as group B Streptococcus (GBS), corresponds to a species of spherical Gram-positive bacteria often organized in pairs or chains. Initially, it was thought that pathogenic infections of S. agalactiae were just present in mammalian species. However, nowadays there are many diseases linked to this pathogen in fishes, reptiles and frogs. Also, S. agalactiae is usually found in the free environment, such as in milk and dairy herds’ feces. Although this species is frequently associated with the normal nonvirulent human microbiota, it can also be related to severe diseases in its invasive form. The cell wall of pathogenic bacteria is normally associated with many virulence factors, and, besides the capsule, flagella and pilus, Gram-positive bacteria also have many surface proteins responsible for recognizing and binding to eukaryotic molecules, such as adhesins, toxins, proteases, and invasins. Two bacterial adhesins with notable influence in S. agalactiae virulence are the serine rich repeat glycoproteins Srr1 and Srr2. These proteins are known to interact with human fibrinogen and plasminogen. Importantly, Srr2 has a higher affinity to fibrinogen compared with Srr1 and is only associated with S. agalactiae ST-17 hypervirulent isolates. Considering this, the main focuses of this work were referred to the characterization of the interaction between Srr2 and human fibronectin, a big glycoprotein present in the extracellular matrix and in the blood plasma, important for wound healing, cell adhesion and migration. The experiments chosen to characterize the interaction between Srr2 and fibronectin were based on the use of biochemical techniques, such as ELISA, western blot, dot blot, affinity chromatography, BCA protein assay, and SDS-PAGE. Through a comparison between ST-17 and non-ST-17 S. agalactiae strains, it was identified that Srr2 promotes specific binding of ST-17 strains to fibronectin. By using a recombinant Srr2 binding region (rSrr2-BR) produced through affinity chromatography, it was possible to evaluate that the binding affinity between rSrr2-BR and fibronectin differs when using immobilized rSrr2-BR or immobilized fibronectin. In this sense, it is feasible to hypothesize that some specific conformational changes of one or both proteins can be involved in their binding affinity. Furthermore, through western blot and dot blot analysis, it was shown that the binding site of rSrr2-BR on fibronectin is probably represented by its cell-binding domain (CBD) fragment. Moreover, ELISA assays conducted applying different concentrations of NaCl revealed that ionic forces play an important role in the interaction of Srr2 with fibronectin, as already shown to occur with the binding of Srr2 with fibrinogen. Finally, the evaluation of the interaction of mutant forms of rSrr2-BR with fibronectin allowed the identification of the dock, lock and latch (DLL) binding mechanism for this interaction, as previously characterized for Srr2 binding to fibrinogen as well. In conclusion, this study promotes the characterization of a new important interaction between S. agalactiae, an emerging pathogen, and human fibronectin, revealing valuable information surrounding this topic. Studies conducted investigating protein-protein interactions are extremely important for the future development of new drugs and vaccines. Knowing this, through the analysis of the Srr2 structure and its interaction with fibronectin, this virulence factor can be considered a promising candidate for drug targeting as it is exclusively associated with hypervirulent strains resistant to many antimicrobial agents. These S. agalactiae strains represent a huge economic and health care system problem nowadays. So, by focusing research on these topics, we can mitigate the spread of S. agalactiae and the future consequences related to this issue.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28502