The Antioxidative Activity Of Mycoplasma Pneumoniae MsrA And Its Regulation On The Production Of Pro-inflammatory Cytokines And Reactive Oxygen Species When Stimulated With LAMPs

Background:Mycoplasma pneumoniae,an atypical bacterium,is the common pathogenic bacterium responsible for community-acquired pneumonia.M.pneumoniae can adhere to the respiratory epithelial cells,colonize and multiply on them.A series of reactive oxygen species（ROS）,such as Hydrogen peroxide,singlet oxygen and superoxide,was produced by M.pneumoniae through glycerol metabolism.Lipid-associated membrane proteins（LAMPs）extracted from M.pneumoniae can activate macrophages to secrete pro-inflammatory cytokines such as tumour necrosis factorα（TNF-α）,interleukin-1β（IL-1β）,and interleukin-6（IL-6）,and produce ROS to induce a severe inflammation response.The oxidative stress and inflammation response stimulated by M.pneumoniae not only can result in both structural and functional deterioration of respiratory epithelial cells and cilia but also can hurt M.pneumoniae itself.So M.pneumoniae should have some antioxidant and anti-inflammation systems to protect it against oxidative damage and inflammation reaction.Methionine（Met）is one of the essential amino acids in organisms,and it is also the most easily oxidized amino acid.The oxidation of Met leads to two different sulfoxides,namely Met-S-O and Met-R-O,which are stereo-isomers.Methionine sulfoxide reductases（Msr）,which specifically reduce the oxidized methionine（Met-O）to Met through a reduction reaction.Methionine sulfoxide reductase A（Msr A）,which exists in most living organisms,can reduce Met-S-O to Met so as to repair the oxidized proteins,and it also has roles in the detoxification of ROS.Msr A activity has been shown to be important in resisting oxidative stress in many bacteria,such as H.pylori,M.genitalium,E.coli and M.tuberculosis.In addition,Msr A has also been reported to be an important virulence factor in some pathogenic bacteria（for example,E.coli,M.genitalium,S.pneumoniae,etc.）,which can affect a range of properties like adherence,motility,in vivo survival,intracellular survival and biofilm formation.Furthmore,Msr A of M.genitalium can inbibit the secretion of TNF-α,IL-1βin THP-1 and He La cells.Objective:M.pneumoniae Msr A is a 17 KD protein,encoded by mpn607/msr A.This study intends to assay the methionine sulfoxide reductase activity and key sites of enzymatic activity of M.pneumoniae Msr A,and to investigate the effect of M.pneumoniae Msr A to produce the TNF-α,IL-1β,IL-6 and ROS in LAMPs-treated THP-1 cells.This work will enrich the antioxidant and anti-inflammatory systems of M.pneumoniae,and help further understand the immune evasion mechanism of M.pneumoniae and the interaction between this pathogen and host cells.Methods:1.According to the msr A gene sequence from Genbank,msr A DNA sequence was synthesized,and a prokaryotic expression recombinant plasmid p ET28a（+）-msr A was constructed.To express the recombinant protein Msr A（r Msr A）,plasmid p ET28a（+）-msr A was transform to the expression strain E.coli BL21（DE3）,and induce expression by IPTG.After the recombinant protein was identified by Western blot,it was purified by Ni²⁺-NTA affinity chromatography,and then the protein was purified and concentration was determined by BCA kit.The enzymatic activity of r Msr A to reduce Met-S-O to Met was determined spectrophotometrically based on DTNB-DTT assay.2.According to the literature and bioinformatics analysis,the enzymatic active key site of Msr A（the 10th Cys）was predicted.Two mutant msr A genes were synthesized so as the 10th Cys change to Ser or Ala in M.pneumoniae Msr A protein.The recombinant plasmids p ET28a（+）-msr A-S₁₀and p ET28a（+）-msr A-A₁₀were constructed,and the recombinant proteins r Msr A-S₁₀and r Msr A-A₁₀were expressed,identified and purified.The concentrations of these two proteins were determined by BCA kit.Enzymatic activities of r Msr A-S₁₀and r Msr A-A₁₀to reduce Met-S-O to Met were determined spectrophotometrically based on DTNB-DTT assay.3.The purified r Msr A was intramuscularly injected into6-week-old BALB/c female mice with Freund’s adjuvant.2 weeks after the last immunization,blood was collected,and serum was separated,and the antibody titer was detected by ELISA.M.pneumoniae in log phase was stimulated with different concentrations of hydrogen peroxide（H₂O₂）or tert-Butysl hydroperoxide（t-BHP）,then the m RNA transcription level of msr A gene was analyzed by reverse transcription-PCR（RT-PCR）,and the protein expression of M.pneumoniae Msr A was detected by Western blot.4.LAMPs were extracted from M.pneumoniae,and BCA kit was used to determine its concentration.THP-1 cells were pre-treated with different concentrations of r Msr A for a different time before exposure to5μg/m L LAMPs for 24 h,and then intracellular ROS was determined using ROS detection kit.THP-1 cells were pretreated with 30μg/m L r Msr A for 1 h prior to exposure to 5μg/m L LAMPs for 24 h,and intracellular SOD was assayed by kit and TNF-α,IL-1βand IL-6 in the cell supernatant were detected by ELISA.5.THP-1 cells were pre-treated with 30μg/m L r Msr A for 1 h prior to exposure to 5μg/m L LAMPs for 24 h.The translocation of NF-κB in cells was observed by confocal microscopy after indirect immunofluorescence,p-p65 and p65 also be detected by Western blot.Result:1.The p ET28a（+）-msr A prokaryotic expression recombinant plasmid was constructed.A soluble fusion protein with a relative molecular weight（Mr）of about 17 k D was expressed and was identified as r Msr A by Western blot.The activities of 10μg,15μg and 20μg purified r Msr A to convert Met O into Met were much higher than heat-inactivated r Msr A（t=26.39,P<0.01;t=57.28,P<0.01;t=7.905,P<0.05）,and the enzymatic activity was highest when stimulated with 15 or 20μg purified r Msr A.2.The key amino acid which impacts on the enzymatic activity of Msr A was predicted to be Cys10 according to literature and bioinformatics.Genes msr A-S₁₀and msr A-A₁₀were synthesized,and the prokaryotic expression recombinant p ET28a（+）-msr A-S₁₀and p ET28a（+）-msr A-A₁₀were successfully constructed.r Msr A-S₁₀and r Msr A-A₁₀were expressed and purified.The activities of r Msr A-S₁₀and r Msr A-A₁₀to converting Met O into Met were much lower than r Msr A（t=3.868,P<0.01;t=3.807,P<0.01）.There was no significant difference in the enzymatic activity between r Msr A-S₁₀and r Msr A-A₁₀（t=0.330,P>0.05）.3.When M.pneumoniae were treated with 4mmol/L H₂O₂or2mmol/L t-BHP,M.pneumoniae msr A gene m RNA transcription levels and the expression of Msr A protein were much higher than PBS treated group（t=5.544,P<0.05;t=12.75,P<0.01）.4.When THP-1 cells were being pretreated with 10～50μg/m L r Msr A before explor to M.pneumoniae LAMPs,the intracellular ROS were much lower than cells being pretreated with PBS,the content of intracellular ROS reduced to the lowest when THP-1 cells were being pretreated with 30μg/m L r Msr A.When being pretreated with r Msr A for0.5～4h,the intracellular ROS in M.pneumoniae LAMPs stimulated THP-1 cells were decreased than no Msr A pretreated cells,the level of ROS reduced to the lowest when THP-1 cells were being pretreated with30μg/m L r Msr A for 1h.When THP-1 cells were pretreated with 30μg/m L r Msr A for 1 h prior to exposure to 5μg/m L LAMPs for 24 h,the SOD in cells was increased（t=8.24,P<0.05）,while TNF-α,IL-1βand IL-6 in the cell supernatant were decreased when comparing with no r Msr A pretreated cells（t=8.21,P<0.05;t=5.10,P<0.05;t=12.87,P<0.01）.5.M.pneumoniae LAMPs were used to stimulate THP-1 cells pretreated with M.pneumoniae Msr A,there was a significant increase in the expression of phosphorylated NF-κB p-p65 in the r Msr A pretreatment THP-1 cells compared with that of PBS pretreatment cells when stimulated with LAMPs（t=5.362,P<0.05）,and the translocation of NF-κB from cytoplasm to nucleus was inhibited when compared with LAMPs stimulated cells with PBS pretreatment.Conclusions:1.The expression of M.pneumoniae Msr A was up-regulated when stimulated with H₂O₂and t-BHP,r Msr A has an antioxidative activity by reducing Met-O to Met,and Cys10 was the key amino acid which impact on the enzymatic activity of Msr A.2.M.pneumoniae Msr A can inhibited the NF-κB signal transduction pathway and reduced the production of ROS,TNF-α,IL-1βand IL-6 in M.pneumoniae LAMPs-stimulated THP-1 cells.