Inhibitory Effect Of Rutin On Sortase A Activity Produced By Streptococcus Agalactiae

Streptococcus agalactiae(S.agalactiae),also known as the group B streptococcus,is an important pathogen that can cause diseases in human,animals,and a variety of aquatic species.Streptococcus agalactiae is main pathogen that responable for disease of tilapia in aquaculture.Antibiotics are the main approach of treatment infections for tilapia caused by S.agalactiae.However,due to the abuse of antibiotics,S.agalactiae has developed serious resistance,resulting in the decline or treatment failure of antibiotics,which seriously threaten the healthy development of tilapia farming.Therefore,it is urgent to develop novel mechanisms of anti-tilapia S.agalactiae infection drugs.Chinese herbal medicines have been used for thousands of years in China,with the advantages of a wide range of sources,low or no toxicity,and many active substances,Chinese herbal medicine has become the first choice to alternative antibiotics against bacterial infections.Sortase A(Srt A)is a transpeptidase,secreted by most Gram-positive bacteria,and is related to the process of bacterial adhesion to the surface of host cells.In this paper,Srt A was used as target to investigate the effect of rutin on the pathogenicity of S.agalactiae in vitro.The results are as follows:1.In this paper,the recombinant Srt A was expressed and purified,then the activity of purified Srt A was determined.The results showed that a large amount of soluble protein will obtained after inducted at 37 ? with 1 mmol/L IPTG for 6 h.Chromatography and ion exchange chromatography were performed to obtain the Srt A with purity greater than 85%.Purified Srt A could significantly increase the fluorescence intensity of the reaction system,indicating that Srt A had biological activity.2.The fluorescence resonance energy transfer(FRET)method was used to determine the inhibitory effect of natural compound rutin on Srt A activity,the experiment further determined the effect of rutin on S.agalactiae,through testing the minimum inhibitory concentration(MIC),growth curves,adhesion to human fibronectin and the effect on biofilm formation.The results showed that the MIC of rutin agianst S.agalactiae was higher than 256 ?g/m L,rutin at concentrations between0-256 ?g/m L had no influenc on the growth of S.agalactiae.The adhesion of S.agalactiae to human fibronectin was significantly inhibited by rutin at concentrations higher than 16 ?g/m L.Moreover,the biofilm formation was reduced statistically by addition of rutin higher than 8 ?g/m L.3.Polyclonal antibodies aiganst Srr1 were prepared to detect the ancoring of Srr1 on surface of S.agalactiae.Dot-blot results showed that the anchorage of Srr1 on the surface of S.agalactiae decreased at a dose dependent manner.The results showed that rutin could reduce the anchorage of Srr1 on the surface of Streptococcus agalactiae by inhibiting Srt A activity.In summary,rutin can inhibit the activity of Srt A without inhibiting the growth of S.agalactiae;reduce the adhesion of S.agalactiae to human fibronectin and the formation of biofilm;reduce the virulence protein Srr1 anchoring on the surface of S.agalactiae.This study suggests that rutin can be used as an effective drug for the treatment of S.agalactiae infection on tilapia.