Transcriptional Regulation Of Virulence Factors Spa And ClfB By The SpoVG-Rot Cascade In Staphylococcus Aureus

Staphylococcus aureus is a community-and hospital-acquired bacterial pathogen that can cause both local and systemic infections in humans,such as sepsis,endocarditis,and pneumonia.The abilities of S.aureus to colonize a wide range of organs and to cause acute and chronic diseases are mainly due to the large arsenal of virulence factors such as toxins,proteases,nucleases,lipases,and adhesive proteins.The production of virulence factors in S.aureus is controlled by a network of interacting regulators,mainly including two-component systems,sigma factors,regulatory proteins,and regulatory RNAs.The SarA family,a group of proteins homologous to the virulence global regulator SarA,is one of the foremost families of global regulators in S.aureus.Rot,the single domain protein of the SarA protein family,can regulate the expression of numerous viulence genes.SpoVG was originally identified as a factor involved in the sporulation and asymmetric cell division in Bacillus subtilis.In S.aureus,SpoVG influences the production of capsule,extracellular nuclease,protease,and lipase by regulating the transcription of cap,nucl,splE,and lip.Moreover,SpoVG is involved in methicillin and glycopeptide resistance of methicillin-resistant S.aureus(MRSA)and vancomycin-intermediate S.aureus(VISA)by regulating the expression of cell wall-associated genes.This dissertation for doctor’s degree mainly explored the molecular mechanism of SpoVG regulation through the following two parts of study:Firstly,we found that the transcription level of rot was significantly decreased in the S.aureus spo VG mutant strain compared with that of the wild-type strain.The rot promoter-lacZ fusion reporter assay showed that the promoter activity of rot decreased in the spo VG mutant strain compared with that in the WT strain by the P-Galactosidase activity assay,suggesting that SpoVG is a positive regulator of rot.In addition,the electrophoretic mobility shift assay showed that SpoVG was able to bind to the rot promoter specifically.The S.aureus Sar-family regulatory protein Rot has been reported to regulate many important extracellular virulence factors at the transcription level,including protein A and clumping factor B.By introducing the spo VG rot double mutant,we demonstrated that SpoVG played multiple regulatory roles in the transcription of spa and clfB through direct-binding to the respective promoters and in a Rot-mediated manner.Our results further found that the spoVG mutant strain displayed significantly decreased fibrinogen-binding and host cell infection abilities compared with the WT strain.Moreover,we also found that the Stkl-mdieated phosphorylation of SpoVG could promote its binding to the promoters of rot,spa,and clfB.Collectively,our data revealed a novel SpoVG regulatory cascade in S.aureus and the molecular mechanisms which SpoVG is involved in the regulation of cell surface protein Spa and ClfB.S.aureus aggregation is a biological process through which cells bind matrix proteins and form stable clumps to evade host defenses and to adapt to antibiotic stress.In aggregate communities,S.oaureus adjust the distribution of its adhesins and surface proteins to promote its tolerance to hazardous environments.We noticed that spo VG mutant strain displayed a severe cell aggregation which dispeared after digestion with protease,suggested that surface proteins maybe involved in the aggregation formation.RT-qPCR result showed that the transcription level of sasC was significantly decreased in the S.aureus spo VG mutant strain compared with that of the wild-type strain.SasC,one of S.aureus surface adhesins,is reported to involved in the cell aggregation and biofilm accumulation.No obvious cell aggregation was observed when the sasC aggregation region was deleted in the spoVG mutant strain,indicated that the SasC aggregation domain is involved in the cell aggregation phenotype of spoVG mutant strain.Moreover,fast protein liquid chromatography(FPLC)coupled with multi-angle light scattering(FPLC-MALS)analysis result revealed that SasC can form a superpolymer.Electrophoretic mobility shift assay result demonstrated that SpoVG can bind to the promoter region of sasC specifically.These results indicate that SpoVG may modulate the cell aggregation ability of S.aureus by regulating sasC gene expression.Collectively,we have found a novel SpoVG-Rot regulatory cascade in S.aureus and the molecular mechanism that SpoVG-Rot cascade regulates spa and clfB expression has been studied.The study of novel regulatory cascades can facilitate us to reach a deeper understanding of the regulatory patterns of gene expression involved in virulence and pathogenicity of S.aureus.