Streptococcus agalactiae is an opportunistic pathogen colonizing the genital and gastrointestinal tracts of healthy adults. Its transmission from a colonized mother to her infant leads to invasive neonatal infections, with severe associated symptoms, leading also to death. In addition, S. agalactiae may cause diseases in elderlies an immunodeficient subjects. Despite many S. agalactiae virulence factors are known, the regulatory mechanisms underlying pathogenesis are not well characterized yet. The transcriptional regulator CodY regulates gene expression in almost all low G+C Gram-positive bacteria. Its activity is dependent on the availability of two cofactors: the branched-chain amino acids and GTP. In pathogens, this protein is involved in both the control of the metabolism and pathogenesis. The role of CodY in the regulation of gene expression and virulence in S. agalactiae is unknown. Understanding the molecular mechanisms controlling the onset of pathogenesis may allow the development of new therapeutic strategies. In this context, this work was aimed at studying the role of the global transcriptional regulator CodY in S. agalactiae, particularly in the hypervirulent BM110 strain. To this purpose, a codY deletion mutant of the BM110 strain was prepared and named BM110ΔcodY. The obtained mutant strain showed no growth defects compared to the wild-type. In order to determine whether the deletion of codY affected regulation of gene expression in BM110, the level of expression of two genes was determined. During exponential phase in rich medium, the genes ilvE and livK were expressed at higher levels in the deletion mutant compared to the wild-type, demonstrating the role of CodY as a transcriptional repressor. In order to determine the CodY regulon in S. agalactiae, RNA-Seq experiments were performed. The sequencing results are being analysed. The first process involved in bacterial pathogenesis is the adhesion to the host. The ability of the two strains to adhere to extracellular matrix and plasma components was assessed. In particular, BM110ΔcodY displayed a higher ability to bind to fibrinogen compared to the parental strain. Since FbsA is the major fibrinogen-binding protein in S. agalactiae, its protein and transcript abundance in the two strains was measured. During the exponential phase, the deletion mutant showed a statistically significant small increase in FbsA expression compared to the wild-type bacterium. At the transcript level, although the BM110ΔcodY showed a higher level of expression of the fbsA gene, the difference was not statistically significant. Since no putative CodY binding sites can be identified upstream of fbsA, it is likely that the observed slight increase in fbsA transcript and protein levels is the result of an indirect effect of codY deletion. Notably, the small increase in FbsA levels in BM110ΔcodY during the exponential phase could hardly explain the significant increased adhesion to fibrinogen by this strain. The higher ability of the codY mutant to bind to fibrinogen may therefore be ascribed to an altered expression of other adhesins involved in fibrinogen-binding. The results of the RNA-Seq analysis will allow the detection of all the adhesins involved in fibrinogen-binding which are regulated by CodY. As a final analysis, the effect of codY deletion on the ability of BM110 to adhere to epithelial cells was assessed by adhesion assays on an A549 lung epithelial cells monolayer. The deletion mutant showed a significant increment in the adhesion to A549 cells compared to the wild-type strain, suggesting a role for CodY in the control of cell adhesion. The increased ability of the mutant strain to adhere to extracellular matrix and plasma components, as well as to host cells, suggests that CodY may be involved in the control of the colonization process. As a consequence, we may speculate that CodY plays a role in controlling the virulence of S. agalactiae.
S. agalactiae colonizza il tratto genitale e gastrointestinale di adulti sani. La sua trasmissione verticale da madre a figlio può essere causa di infezioni neonatali invasive, causando anche la morte. Inoltre, S. agalactiae può causare malattie nei soggetti anziani ed immunodeficienti. Nonostante siano noti molti fattori di virulenza di S. agalactiae, i meccanismi regolatori alla base della patogenesi non sono ancora chiari. Il regolatore trascrizionale CodY regola l'espressione genica in quasi tutti i batteri G+ a basso contenuto in G+C. La sua attività dipende dalla presenza di aminoacidi a catena ramificata e GTP. Nei batteri patogeni, questa proteina è coinvolta sia nel controllo del metabolismo che nella patogenesi. Il ruolo di CodY nella regolazione dell'espressione genica in S. agalactiae è sconosciuto. In questo contesto, questo lavoro era finalizzato allo studio del ruolo di CodY in S. agalactiae, in particolare nel ceppo ipervirulento BM110. A tale scopo è stato preparato un mutante di delezione del gene codY nel ceppo BM110, chiamato BM110ΔcodY. Il mutante ottenuto non ha mostrato difetti di crescita rispetto al wild-type. Al fine di determinare se la delezione di codY ha influenzato la regolazione dell'espressione genica in BM110, è stato determinato il livello di espressione di due geni. Durante la fase esponenziale di crescita, i geni ilvE e livK sono stati espressi a livelli più elevati nel mutante rispetto al wild-type: CodY agisce come repressore trascrizionale. Al fine di determinare il regulone di CodY in S. agalactiae, sono stati condotti esperimenti di RNA-Seq. I risultati del sequenziamento sono in fase di analisi. Il primo processo coinvolto nella patogenesi batterica è l'adesione all'ospite. È stata valutata la capacità dei due ceppi di aderire alle componenti della matrice extracellulare e del plasma. In particolare, BM110ΔcodY ha mostrato una maggiore capacità di legare il fibrinogeno rispetto al wild-type. Poiché FbsA è la principale proteina legante il fibrinogeno in S. agalactiae, la sua abbondanza a livello di proteina e trascritto è stata valutata nei due ceppi. Durante la fase esponenziale di crescita, il mutante ha mostrato un piccolo aumento statisticamente significativo nell'espressione di FbsA rispetto al wild-type. A livello di trascrizione, sebbene BM110ΔcodY mostrasse un livello più alto di espressione del gene fbsA, la differenza non era statisticamente significativa. Poiché non è possibile identificare siti putativi di legame di CodY a monte di fbsA, è probabile che il leggero aumento nei livelli di trascritto e proteina sia il risultato di un effetto indiretto della delezione di codY. Inoltre, il piccolo aumento dei livelli di FbsA in BM110ΔcodY durante la fase esponenziale di crescita potrebbe difficilmente spiegare il significativo aumento dell'adesione al fibrinogeno da parte di questo ceppo. La maggiore capacità del mutante di legare il fibrinogeno può quindi essere attribuita ad un'espressione alterata di altre adesine leganti fibrinogeno. I risultati di RNA-Seq consentiranno il rilevamento di tutte le adesine leganti fibrinogeno che sono regolate da CodY. Come ultima analisi, l'effetto della delezione di codY sulla capacità di BM110 di aderire a cellule epiteliali è stato valutato mediante saggi di adesione su un monostrato di cellule epiteliali polmonari (A549). Il mutante ha mostrato un aumento significativo dell'adesione alle cellule rispetto al wild-type, suggerendo un ruolo per CodY nel controllo dell'adesione cellulare. L'aumentata capacità del mutante di aderire alle componenti della matrice extracellulare e del plasma, nonché alle cellule ospiti, suggerisce che CodY può essere coinvolto nel controllo del processo di colonizzazione, e che quindi abbia un ruolo nel controllo della virulenza di S. agalactiae.
Deletion of the gene encoding the transcriptional regulator CodY and analysis of its role in the control of cell adhesion in Streptococcus agalactiae
BONACORSI, ANDREA
2019/2020
Abstract
Streptococcus agalactiae is an opportunistic pathogen colonizing the genital and gastrointestinal tracts of healthy adults. Its transmission from a colonized mother to her infant leads to invasive neonatal infections, with severe associated symptoms, leading also to death. In addition, S. agalactiae may cause diseases in elderlies an immunodeficient subjects. Despite many S. agalactiae virulence factors are known, the regulatory mechanisms underlying pathogenesis are not well characterized yet. The transcriptional regulator CodY regulates gene expression in almost all low G+C Gram-positive bacteria. Its activity is dependent on the availability of two cofactors: the branched-chain amino acids and GTP. In pathogens, this protein is involved in both the control of the metabolism and pathogenesis. The role of CodY in the regulation of gene expression and virulence in S. agalactiae is unknown. Understanding the molecular mechanisms controlling the onset of pathogenesis may allow the development of new therapeutic strategies. In this context, this work was aimed at studying the role of the global transcriptional regulator CodY in S. agalactiae, particularly in the hypervirulent BM110 strain. To this purpose, a codY deletion mutant of the BM110 strain was prepared and named BM110ΔcodY. The obtained mutant strain showed no growth defects compared to the wild-type. In order to determine whether the deletion of codY affected regulation of gene expression in BM110, the level of expression of two genes was determined. During exponential phase in rich medium, the genes ilvE and livK were expressed at higher levels in the deletion mutant compared to the wild-type, demonstrating the role of CodY as a transcriptional repressor. In order to determine the CodY regulon in S. agalactiae, RNA-Seq experiments were performed. The sequencing results are being analysed. The first process involved in bacterial pathogenesis is the adhesion to the host. The ability of the two strains to adhere to extracellular matrix and plasma components was assessed. In particular, BM110ΔcodY displayed a higher ability to bind to fibrinogen compared to the parental strain. Since FbsA is the major fibrinogen-binding protein in S. agalactiae, its protein and transcript abundance in the two strains was measured. During the exponential phase, the deletion mutant showed a statistically significant small increase in FbsA expression compared to the wild-type bacterium. At the transcript level, although the BM110ΔcodY showed a higher level of expression of the fbsA gene, the difference was not statistically significant. Since no putative CodY binding sites can be identified upstream of fbsA, it is likely that the observed slight increase in fbsA transcript and protein levels is the result of an indirect effect of codY deletion. Notably, the small increase in FbsA levels in BM110ΔcodY during the exponential phase could hardly explain the significant increased adhesion to fibrinogen by this strain. The higher ability of the codY mutant to bind to fibrinogen may therefore be ascribed to an altered expression of other adhesins involved in fibrinogen-binding. The results of the RNA-Seq analysis will allow the detection of all the adhesins involved in fibrinogen-binding which are regulated by CodY. As a final analysis, the effect of codY deletion on the ability of BM110 to adhere to epithelial cells was assessed by adhesion assays on an A549 lung epithelial cells monolayer. The deletion mutant showed a significant increment in the adhesion to A549 cells compared to the wild-type strain, suggesting a role for CodY in the control of cell adhesion. The increased ability of the mutant strain to adhere to extracellular matrix and plasma components, as well as to host cells, suggests that CodY may be involved in the control of the colonization process. As a consequence, we may speculate that CodY plays a role in controlling the virulence of S. agalactiae.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/20.500.14239/11895