Regulatory Mechanisms Of The Glutamate Decarboxylase System GadT2/gadD2 Mediated Acid Resistance In Listeria Monocytogenes

Listeria monocytogenes（L.monocytogenes,LM）is a gram-positive,facultative anaerobic non-bacillus bacillus,which can survive in extremely harsh environments,such as wide acid,high temperature and high salinity,so it exist widely in the natural environment Medium,and easily contaminate foods such as vegetables,fruits,meat,eggs and seafood.L.monocytogenes can infect humans and a variety of animals.It is an important zoonotic disease.It mainly infects newborns,the elderly,pregnant women,and people with weakened immunity.It’s able to cause meningitis,sepsis and abortion in pregnant women,although the incidence is not so high,but the fatality rate is as high as30%,much higher than common food-borne pathogens.L.monocytogenes is neutrophilic,so acidic stress is the most commonly encountered adverse environmental factors,including:the acidic environment of food processing,the host’s digestive tract,and the acidic environment of the host cell’s phagocytosis.Therefore,resistance to acid stress is simple the prerequisite for successful establishment of infection by L.monocytogenes.In order to better adapt to the acidic environment,L.monocytogenes has multiple anti-acid stress systems,namely the stress regulator SigB（σB）,the arginine deiminase（ADI）system,the agmatine deiminase（AgDI）system and glutamate decarboxylase（GAD）system.The GAD system is ubiquitous in various bacteria.L.monocytogenes usually encodes 2-3 sets of glutamate decarboxylase,but it is still unclear about the anti-acid stress effect of each component in the GAD system and its regulatory mechanism.This study aims to:（1）compare the contribution of each component of the GAD system of L.monocytogenes to the bacteria’s acid stress resistance;（2）screen and clarify the regulatory effects of transcription factors on the GAD system gadT2/gadD2;（3）Prove the molecular mechanism of transcription factors regulating gadT2/gadD2 to mediate the anti-acid stress effect of L.monocytogenes.1.Comparison of the anti-acid stress effects of each acid resistant system in L.monocytogenes.The anti-acid stress effects of the main acid resistant system of L.monocytogenes were compared by survival test.The results showed that the deletion strainΔgadD2 hadthe lowest survival rate in BHI at pH 2.5 and artificial gastric juice,followed byΔsig B,ΔgadD3,Δagu A1 andΔarc A;Compared with the parent strain 10403S,the survival rate ofΔgadD1 andΔagu A2 has no significant difference.The GAD system is essential for the anti-acid stress effect of L.monocytogenes 10403S,and gadD2 plays the main anti-acid stress effect.Analyzing the transcription levels of each component of the GAD system of L.monocytogenes 10403S in the logarithmic phase and the plateau phase,and found that the transcription level of gadT2/gadD2 was significantly higher than that of gadD3 and gadD1/gadT1,whether in the logarithmic or plateau phase.,Indicating that the transcription level of gadT2/gadD2 is positively correlated with its anti-acid stress effect.In order to further study the anti-acid stress effect of gadT2/gadD2 and its regulatory mechanism,this study used homologous recombination technology to successfully knock out the gadT2/gadD2 gene cluster;use a prokaryotic expression vector to express GadD2 protein;use purified GadD2 to prepare the corresponding poly Cloned antibodies.Growth curve and acid stress survival test showed that the growth ability of the deletion strainΔgadT2/gadD2 under sublethal acidic conditions（pH 4.5）was significantly lower than that of the parent strain 10403S;the survival rate under lethal acid stress conditions（pH 2.5）was also significantly lower than the parent strain 10403S,showing that gadT2/gadD2 in L.monocytogenes 10403S plays an important role in resisting acid stress.2.Molecular mechanism of L.monocytogenes GadR4 regulating gadT2/gadD2In Lactococcus lactis,there is a regulatory gene gadR before the promoter of the GAD system（gadC/gadB）gene cluster.The Rgg family transcription regulator GadR encoded by this gene may be involved in regulating the expression of gadC/gadB.Analysis of the L.monocytogenes genome revealed that the structure of the gadT2/gadD2gene cluster was different from that of Lactococcus lactis gadR/gadC/gadB,and there were no regulatory genes nearby;however,the genome of L.monocytogenes 10403S did have 5 Rgg family transcriptional regulation Factor-encoding genes are distributed in 4positions in the genome and are named gadR1,gadR1’,gadR2,gadR3,and gadR4.Analysis of the amino acid homology of GadR between L.monocytogenes and Lactococcus lactis found that GadR4 of L.monocytogenes has the highest homology with GadR of Lactococcus lactis（31.8%）.Therefore,this study speculates that GadR4 may be involved in the regulation of gadT2/gadD2.In this study,a gadR4 gene deletion strain（ΔgadR4）was constructed using homologous recombination technology.The acid stress survival test showed that the survival rate ofΔgadR4 was significantly lower than that of the parent strain 10403S after treated with BHI at pH 2.5 for 1 hour.Western blot results showed that under different pH conditions,the expression level of GadD2 inΔgadR4 was significantly higher than that of the parent strain 10403S.The cell invasion test showed that the invasion rate ofΔgadR4 on gastric cancer cell MGC803 was significantly lower than that of the parental strain.But mouse experiments showed that 24 hours after intraperitoneal injection infection,the bacterial loadin the liver and spleen of mice in theΔgadR4 infection group was significantly higher than that of the parent strain 10403S.It is suggested that GadR4 may mediate the resistance to acid stress and pathogenicity of L.monocytogenes by regulating gadT2/gadD2.3.Molecular mechanism of L.monocytogenes GadR4 regulating gadT2/gadD2In order to explore the molecular mechanism of GadR4 regulating gadT2/gadD2 to mediate the resistance to acid stress and pathogenicity of L.monocytogenes,this study constructed a complement strain（CΔgadR4）and gadT2/gadD2 GFP reporter plasmid(P_gadT2-gfp).The reporter plasmid can be expressed in the parent strain 10403S,the deletion strainΔgadR4 and the complementing strain CΔgadR4.After treated with different pH conditions,the fluorescence intensity ofΔgadR4 was significantly higher than that of the parent strain,while the fluorescence intensity of CΔgadR4 was comparable to that of the parent strain.Western blot results also showed that the absence of gadR4 significantly up-regulated the expression level of gadD2,suggesting that GadR4is involved in the negative regulation of gadT2/gadD2 expression.The prokaryotic expression of GadR4 was further obtained,and the purified GadR4 was used for aggregation and migration retardation test.As a result,it was found that GadR4 could not bind to the promoter P_gadT2 of gadT2/gadD2.It suggests that GadR4 may not directly regulate gadT2/gadD2 to mediate the anti-acid stress and pathogenicity of L.monocytogenes.In summary,this study found that the gadT2/gadD2 of the GAD system of L.monocytogenes 10403S contributes the most to the acid stress resistance of the bacteria;the Rgg transcription regulator GadR4 mediates the acid stress resistance and pathogenicity of L.monocytogenes;GadR4 participates in the negative regulation of gadT2/gadD2 expression,but it does not directly bind to the gadT2/gadD2 promoter.The research results lay the foundation for in-depth exploration of the anti-acid stress mechanism of L.monocytogenes.