Study On Mechanism Of Cytoprotective Effect Of Streptococcus Thermophiles Against Oxidative Stress

Streptococcus thermophiles is an important industrial strain,widely used in manufacture and fermentation of yogurts and cheeses,and considered as the second most important species after Lactococcus lactis in dairy industry,which was ingested over 1021 live cells annually by the human population.Recently,several beneficial effects of S.thermophilus in the host have already been reported.S.thermophilus in yogurt cultures has been shown to improve lactose digestion in lactose intolerant individuals because it possesses a ?-galactosidase.In addition,yogurt,which is manufactured with S.thermophilus and Lactobacillus bulgaricus,was awarded a health claim in 2010 by EFSA for this beneficial effect.Therefore,several studies have highlighted its survival through the GIT,the enhancement of the intestinal barrier function,and limiting pathogen adhesion and invasion.Among these probiotic functions,the antioxidative effects of S.thermophilus have been most focused.There are many enzymes and active compounds related to antioxdative activities in S.thermophilus,and superoxide dismutase(SOD)is the most clearly elucidated,which is converted superoxide anions to oxygen and peroxide hydrogen and protect cells against oxidative stress.Recent studies identified a novel peroxidase gene(efeB)and a bifunctional glutathione synthetase gene(gshF)in the genome of S.thermophilus.These two genes was able to promote the oxidative tolerance of the strains,suggesting that the enzymes encoded by the genes had the roles of scavenging ROS and defending oxidative stress.However,whether S.thermophilus carried by yogurt in gastrointestinal tract possessed antioxidative activities and exerted the beneficial effects were still limited.This study was conducted to analyze the antioxdative activities and anti-inflammatory effects of the strain and produced peroxidase EfeB and glutathione in vivo and vitro,using S.thermophilus CGMCC 7.179 as the main material and HT-29 cells as a cell model,and explore the regulatory mechanism of global regulator CodY against oxidative stress for S.thermophilus ST2017.The detailed contents and results were as follows:1.In vitro evaluation of antioxidative properties of S.thermophilusS.thermophilus has a long history in yogurt fermentation,and is recognized as"generally recognized as safe"(GRAS)species.To evaluate the antioxidative activities of these strains,and screen the excellent starter strains,in vitro evaluation of antioxidative activities was performed among 42 of S.thermophilus strains isolated from traditional yogurts in Sinkiang,Inner Mongolia of China.The results showed that,all the tested strains showed the certain activities aganist ascorbic acid and DPPH radical,and high capabilities of scavenging superoxide anions and producing SOD,indicating that S.thermophilus possessed antioxidative activities.In addition,dispersion analysis found that,within each group,wide dispersion of the values of antioxidative parameters was observed,suggesting the antioxidative properties of S.thermophilus were strain-specific features.According to the results above,4 strains with high antixidative activity and 3 strains with low antixidative activity were selected to determine the inhibition activities of lipid peroxidation,and 2 strains with high antixidative activity(strain SDMCC050231 and SDMCC050242)also showed the high activities against lipid peroxidation,even higher than 4 of Lactobacillus strains.All the results demonstrated that S.thermophilus possessed antioxidative capabilities,and screened 2 strains with high antixidative activity.2.Cytoprotective effect of S.thermophilus CGMCC 7.179 against oxidative stress on HT-29 cells.S.thermophilus could synthesize various enzymes and compounds related to antioxidative effect,such as SOD and glutathione.It has been reported that S.thermophilus CGMCC 7.179 expressed a nove dye-decolorizing peroxidase EfeB,which was involved in defending oxidative stress and protected strains growing under aerobic conditions.In this study,after co-incubation of HT-29 cells with S.thermophilus CGMCC 7.179,the intracellular ROS content induced by H2O2 reduced 20%,suggesting that S.thermophilus CGMCC 7.179 was able to attenuate the ROS over-production under oxidative stress.Meanwhile,S.thermophilus CGMCC 7.179 obviously promoted the activities of SOD,GSH-px,and CAT in HT-29 cells under H2O2 stimulation,1.4-fold,1.5-fold,and 3-fold higher than that of the efeB mutant treatment,respectively,suggesting that peroxidase EfeB was involved in antioxidative effect of S.thermophilus CGMCC 7.179.Furthermore,the protein content of nuclear factor erythroid 2-related factor 2(Nrf2)in HT-29 cells following strain CGMCC 7.179 treatment was 1.4-fold higher than that of normal cells,and the increased transcription levels of Nrf2-related antioxidant enzyme genes(HO-1,CGLC,NQO1,and TXNRD1)were also observed,confirming the antioxidative mechanism of activation of Nrf2-ARE pathway for S.thermophilus CGMCC 7.179.However,when co-incubation with efeB mutant,the Nrf2 protein content and its related gene transcription levels were not promoted in HT-29 cells,indicating that EfeB played an important role in cellular antioxidative mechanism of S.thermophilus CGMCC 7.179.All these results demonstrated that S.thermophilus CGMCC 7.179 enhanced cellular antioxidative responses and endowed host cells with protective effects against oxidative stress mediated by the peroxidase EfeB.3.Antioxidative effect of peroxidase EfeB for host cellsPeroxidase EfeB is the only secreted protein using the TAT system in lactic acid bacteria.To further study the cellular antioxidative effect mediated by secreted EfeB,efeB of S.thermophilus CGMCC 7.179 was cloned and expressed in E.coli.Purified EfeB protein at different concentrations co-incubated with HT-29 cells for 24 h,and the cell viabilities were determined under 5 mM H2O2.The results showed that.purified EfeB protein at the concentration of 1-1.5 ?g/mL could protect the cells against oxidative stress.When supplemented with 1 ?g/mL EfeB protein,SOD activities of HT-29 cells was 3-fold higher than that of normal cells;meanwhile,EfeB protein promoted the protein content of Nrf2 and transcription level of HO-1 gene,1.2-and 1.6-fold higher than normal cells,respectively,suggesting that the cellular antioxidative effect of EfeB protein is associated with the activation of Nrf2-ARE pathway.Furthermore,the antioxidative effect of efeB mutant exogenously supplemented with EfeB protein on HT-29 cells restored or exceeded the effect of the wild-type strain,demonstrating that extracellular secretion of EfeB protein confered antioxidative effect to cells by S.thermophilus CGMCC 7.179.All these results indicated that peroxidase EfeB with extracellular antioxidant activity was able to protect cells against oxidative stress through activation of Nrf2-ARE pathway,and conferred S.thermophilus with effective antioxidative activities.4.Antli-inflammatory effect of glutathione biosynthesis on S.thermophilus CGMCC 7.179Glutathione(GSH)is an important bioactive compound,widely distributed in living organisms,which possess many beneficial effects,such as antioxidative,anti-inflammatory and detoxifying effects.Glutathione biosynthesis was catalyzed by a novel bifunctional glutathione synthetase(GshF)in S.thermophilus,and played important roles in defending oxidative stresses.In this study,constructed gshF mutant from S.thermophilus CGMCC 7.179 was sensitive to oxidative stress,and the glutathione yield of this mutant was obviously lower than that of wild-type strain.When co-incubation with gshF mutant,the viability of HT-29 cells was much lower than that with strain ST2017 under H2O2 stimulation,suggesting that the oxidative tolerance of HT-29 cells was associated with glutathione biosynthesis catalyzed by GshF.In addition,gshF mutant treatment significantly attenuated the intracellular activities of antioxidant enzymes and transcription levels of Nrf2-related genes,indicating that glutathione biosynthesis has important physiological effects on S.thermophilus.Furthermore,Elisa analysis showed that S.thermophilus CGMCC 7.179 treatment could reduce the production of IL-8 triggered by Salmonella braenderup H9812,and inhibited the transcription levels of pro-inflammatory factors CXCL1 and CCL20.However,compared with stains CGMCC 7.179,gshF mutant treantment had no effect in relieving the production and expression of pro-inflammatory factors,demonstrating that glutathione had important roles in anti-inflammatory activity.All the results suggested that glutathion biosynthesis by GshF could contribute to anti-inflammatory effects of S.thermophilus CGMCC 7.179 on the human intestinal epithelial cells,and was essential for beneficial functions of S.thermophilus.5.Transcriptional regulator CodY controls the oxidative stress response of S.thermophilus ST2017CodYst,a globally transcriptional regulator,modulated the metabolic network in S.thermophilus.The deficiency of codY promoted the transcription level of covR,a environmental factor,and decreased the survival of strain cells,suggesting that CodY was involved in defending oxidative stress.This study was focused on the regulatory mechanism of CodY against oxidative stress in S.thermophilus ST2017.By the analysis of transcriptome and RT-qPCR,the transcriptional level of a bifunctional glutathione synthetase gene(gshF)was down-regulated by about 3-fold in the codYst defective mutant,along with the decrease by 20%of the glutathione yield compared with the wild-type in minimal chemical defined medium,and a half of the viable cells remained under H2O2 challenge.In vitro gel shift assays showed that the purified CodYst could bind to the promoter region of gshF,with a conserved CodYst-box,confirming the regulation of CodYst on the gshF gene,suggesting that CodY was involved in response to oxidative stress mediated by the regulation of gshF gene in S.thermophilus ST2017.Moreover,transcriptome and RT-qPCR analysis also confirmed the regulatory effect of CodY on two component system CovRS.The results showed that covR mutant was sensitive to oxygen.in the environment,indicating the regulatory effect of CovR against oxidative stress.However,the tolerance of covR mutant to H2O2 was elevated in 30 min,but sharply decreased to 0,suggesting that the regulation of CovR on oxidative stress response was not merely a promotion in S.thermophilus ST2017.Transcriptome analysis found that CovR initiated the transcriptional expression of glutaredoxin subunits,thus we speculated that CovR maintained the redox balance by regulating the glutaredoxin transcription in S.thermophilus ST2017.All the results indicated two ways that CodY regulated the response to oxidative stress in S.thermophilus ST2017:directly controlling the glutathione biosynthesis by GshF,and indirectly modulating the expression of glutaredoxin and other regulators by CovR.