The Role Of The Global Transcriptional Regulator CodY In Promoting Listeria Monocytogenes Against Oxidative Stress

Listeria monocytogenes is a foodborne pathogen that causes listeriosis,leading to pregnant women to abort spontaneously,to newborns,the elderly,and other immunocompromised people to have meningitis and septicaemia.It is known that L.monocytogenes has been a serious threat to the food industry due to its ability to survive and grow in a wide range of environmental conditions,such as refrigeration temperature,high salt concentration and oxidative stress.Therefore,it is particularly important to prevent and control L.monocytogenes transmission.H2O2,NaClO and other oxidizing disinfectants are commonly used to prevent bacterial contamination in food processing,but they will result oxidizing environment which can stimulate the oxidative stress mechanisms in bacteria to resist oxidative damage.These mechanisms include the elimination of oxides and the repairment of cell damage.For example,bacteria can product the catalase(CAT),the superoxide dismutase(SOD)and the glutathione(GSH)to eliminate excessive ROS,and some signal molecules like c-di-AMP as well as SOS reaction are involved in the repair process of DNA damage.However,it is a complex process for bacteria to stimulate oxidative stress response,which often requires the regulatory factors.CodY,a global transcriptional regulatory factor in L.monocytogenes,which has been shown to be involved in a variety of life activities such as virulence,flagellum movement,and carbon and nitrogen metabolism,but the role of CodY in L.monocytogenes to withstand oxidative stress has not been reported.In this study,H2O2 was used to put stress on the wild-type strain EGDe and the isogenic CodY deletion strain EGDe?codY at a mid-logarithmic growth stage(OD600 nm=0.65),in order to explore the role of CodY in the process of oxide-eliminating and cell recovery,and its corresponding regulatory mechanisms.Firstly,in order to verify whether CodY was involved in the antioxidant stress process of L.monocytogenes,we observed the difference in the growth characteristics and the antioxidant stress capacity of the two strains under H2O2 stress.Then,the level of CAT,SOD and GSH and the transcriptional expression of their corresponding coding genes were compared between the two strains to explore the effects of CodY on the expression of different antioxidants in L.monocytogenes.After that,we clarified the specific regulation mechanism of CodY on GSH expression according to the binding of upstream promoter of gshF,which is the GSH-coding gene.Finally,we compared the level of the second messenger c-di-AMP in the two strains to preliminarily explore whether the c-di-AMP was involved in the antioxidative stress process of L.monocytogenes and whether its expression was affected by CodY.At the same time,we compared the DNA damage of the two strains and the expression differences of the important genes in SOS response involved in DNA damage repair to explore the role of CodY in cell recovery.The results showed as follows:(1)The MIC and MBC of EGDe?codY were about twice than that of EGDe under H2O2 stress,and the inhibition zone of EGDe?cody showed a significant difference when the concentration of H2O2 was over 20 mmol/L(P?0.01).The growth of EGDte?codY was completely inhibited by 200 mmol/L of H2O2,while that of EGDe was slightly slowed down.200 mmol/L of H2O2 had an adverse effect on the biofilm formation of both two strains.When stress time reached to 20 min,the biofilm formed by EGDe was loosely,while EGDe?codYcould not form a complete biofilm.The above results indicated that the deletion of CodY would weaken the antioxidant capacity of L.monocytogenes,leading the bacteria inhibited or killed by lower concentration of H2O2 and more difficult to form biofilms in the oxidized environment.(2)The CAT activity in both EGDe and EGDe?codY decreased,but the transcription level of this enzyme had no differences in both strains.The SOD activity did not change significantly,but its expression on mRNA level presented significant differences in those two strains(P ? 0.001).Notably,the general trend of transcription and concentration level of the GSH in the two strains were almost the same,which decreased firstly and then rose up slightly following H2O2 stress.Moreover,the transcription level of the GSH in EGDe?codY was always lower than that in EGDe(P ? 0.01).These results indicated that the effect of CodY on the expression of CAT,SOD and GSH was different in the mRNA level and the final substance production level.(3)There is a CodY-box in the upstream promoter region of gshF.In this study,the electrophoretic mobility shift assay(EMSA)and the isothermal titration calorimetry(ITC)were used to detect the binding of CodY to the promoter region of gshF in vitro.The EMSA result showed that only the reaction system contained the CodY and the gshF promoter probe,a clearly and obviously electrophoresis band with slower migration rate could be observed,indicating a strong binding between the two.The ITC result showed that the heat of binding reaction was generated between the two,which also proved that CodY was bound to the CodY-box sequence in the gshF promoter region.The above results indicated that CodY could act on the gshF promoter region,regulating the expression level of GSH in L.monocytogenes directly.(4)Under the oxidation environment,the expression level of the c-di-AMP synthetase gene dacA in EGDe?codY was lower than that of EGDe,while the expression levels of the hydrolase genes pdeA and pgpH relatively higher,which showed the most significant difference in oxidative stress for 10 min(P?0.01).And the concentration of ATP was significantly lower in EGDe?codY(P ?0.001).The above results indicated that the c-di-AMP synthesis would be affected by CodY deletion.C-di-AMP plays an important role in the bacterial signaling pathway of DNA damage repairment,and therefore CodY may indirectly help L.monocytogenes withstand oxidative damage by regulating the synthesis of c-di-AMP at least shown in our study.(5)The GTS of EGDe and EGDe?codY were 93.1%and 80.0%respectively,which suggested that CodY deletion affected the stability of L.monocytogenes genome and the DNA damage was more serious after oxidative stress.At the same time,the expressions of recA,lexA,recR,lmo1302,and lmo1975,which are important for SOS response,were significantly inhibited in EGDe?codY(P ? 0.001),while the expressions of recA,lmo1302 and lmo1975 in EGDe were activated.The expressions of lexA and recR in EGDe were also activated when the H2O2 stress was given up to 10 and 40 min,although there was no obvious difference up to 20 min treatment.The above results showed that the deletion of CodY would significantly affect the genes transcription involving in DNA damage repairment,and the bacteria have got more serious genome damage,which result in the ability of defencing oxidative stress weaker.Above all,the deletion of CodY reduced bacterial oxidative tolerance,growth,CAT activity and concentration of GSH under the H2O2 stress.CodY could act on the gshF promoter and then to control the level of GSH directly.The deletion of CodY also significantly affected the synthesis of signal molecule c-di-AMP in the DNA damage repair pathway,and the expression of some important genes for DNA damage repair were significantly inhibited,resulting in the bacterial genome stability decreased.Our studies indicate that CodY plays an important role in response to oxidative stress in L.monocytogenes,which would provide a theoretical basis for rational use of H2O2 in daily production,as well as the prevention and treatment of listeriosis.