Study On The Activity Of Brucella SahH In Activated Methionine Cycle

Brucellosis is an allergy anthropozoonosis.It is a chronic infectious disease caused by Brucella invading the body,causing many animals to be infected by each other.It is listed as the first class B infectious disease in China's statutory infectious diseases.People infected with brucellosis mainly manifested after fever,joint pain,fatigue,sweating and other symptoms.Animal infection of brucellosis mainly manifested after female abortion,uterine inflammation,arthritis,inflammation and other symptoms.Therefore,it has caused huge economic losses to the breeding industry.Quorum sensing is mediated by small diffusible molecules termed autoinducers that are synthesized intracellularly to coordinate group behavior.The AI signal molecules of the quorum sensing systerm is divided into three kinds:The first category is mainly Acyl-homoserine lactone?AHL?,also named as AI-1,used by gram-negative bacteria;The second category is mainly oligopeptide signaling molecules used by Gram-positive bacteria;The third category is 4,5-dihydroxy-2,3-pentanedione?DPD?derivatives,also known as AI-2?Autoinducer-2?.In contrast to the species-specificity of the first two types of signal molecules,AI-2 can be used for the exchange of information between bacterial populations and is therefore called the"universal language"for bacterial communication.AI-2 is derived from bacterial pfs/luxS activated methionine cycle?AMC?.In addition,sahH activated methionine cycle also exists in bacterium.This pathway is based on the catalyzed of S-adenosyl-L-homocysteine hydrolase?SahH?by one-step reaction of the substrate S-adenosylhomocysteine?S-Adenosylhomocysteine,SAH?directly generates homocysteine?HCY?.So what pathway does Brucella use for AMC metabolism,does it produce AI-2 signaling molecules?In order to answer these questions,the study first performed detection of AI-2 signaling molecules in Brucella.1.Detection of signal molecule AI-2 in Brucella and optimization of detection methodsThe detection of signal molecule AI-2 using the biological luminescence method of V.harveyi BB170 is the most widely used method at present,and the method is simple and quick.However,this method is susceptible to the detection conditions.Therefore,this study first carried out the optimization of detection methods.By analyzing the growth curve of Vibrio harveyi BB170,the best time of culture supernatant collection of test strains,different medias and antibiotics and other influencing factors,a most suitable V.harveyi BB170 bioluminescence detection method was determined at last.The results of this study indicated that the optimal conditions for the detection ofAI-2 signaling molecules of test strains with BB170 are:Use the fresh cultured bacterium of BB170 which attached to OD600=2.0,to detect the AI-2 signal molecules of the strains cultured in the middle growth stage;When the AB culture medium and the culture supernatant were shaked cultured for 4-6 hours,the data obtained when the assay was performed was the best?the BB152 supernatant prepared with the MB-containing Kanamycin was used as a positive control?.The AI-2 assay was performed on culture supernatants of different OD₆₀₀ values of Brucella A19.The results showed that Brucella could not detect the activity of AI-2 signaling molecules,indicating that the sahH activated methionine cycle was used.2.SahH catalytic activity in activated methionine cycle and analysis of its influencing factorsIn order to further investigate the catalytic activity and function of SahH in the activated methionine cycle,we constructed a prokaryotic expression vector for pET28a-sahH in this study.After transformed into the BL21 host strains and be expressed at 28°C,it could be expressed in large quantities of protein SahH in the supernatant.The study on the purification conditions of the protein showed that the elution with the concentration of 100mM-150mM imidazole had the best purification effect.To further investigate the catalytic activity of SahH,The final concentration of 1 mg/mL of SahH was combined with 1 mM of substrate SAH,The test results showed that SahH can catalyze the production of homocysteine at a substrate concentration of 100.2?M,and the detection results of the AI-2 activity showed that there was no AI-2 activity.At the same time,the prokaryotic expression vectors pET28a-luxS and pET28a-pfs of avian pathogenic E.coli constructed in our laboratory were used to express APEC-LuxS and APEC-Pfs respectively as positive controls.The results showed that286.9?M homocysteine can be produced under the same conditions.and the AI-2 activity of the reaction product showed there is AI-2 activity.In order to study the factors influencing the catalytic activity of SahH,the concentration of SahH enzyme,reaction temperature,thermal stability and the effect of coenzyme NAD⁺were investigated.The results showed that at an SAH concentration of 1 mM,when the concentration of SahH was increased from 1 mg/mL to 4 mg/mL,the homocysteine concentration of the catalytic product increased from 26.6 to 150.3?M?465%?,indicating that the SahH enzyme protein concentration has a significant effect on its catalytic activity;After addition of 500?M exogenous NAD⁺,the homocysteine of its catalytic product decreased from 100.2?M to 21.2?M?78.8%?,indicating that the addition of exogenous NAD⁺significantly inhibited the catalytic activity of SahH;The SahH enzyme proteins were treated at different temperatures 0°C,25°C,37°C,42°C,55°C,60°C,65°C,70°C,and 100°C for 10 min,and then the catalytic activity experiments were performed in vitro.The results showed SahH protein was essentially inactivated after 60°C.After 55°C treatment,the concentration of SahH catalytic product decreased from 40.6?M to 26.6?M?34.5%?.3.Effect of protein modification and catalytic active site on the catalytic activity of SahHIn order to further study the effect of protein modification and catalytic active sites on the catalytic activity of SahH,the above-described established reaction system was used to analyze the effect of protein modification and key catalytically active sites of SahH activity.?1?The double expression plasmid pETDuet-gNAD-sahH containing SahH and NAD⁺kinase was successfully constructed,and the recombinant strain BL21?pETDuet-gNAD-sahH?containing the above plasmid was induced to express SahH successfully.The in vitro catalytic reaction of SahH showed that when the NAD⁺kinase and SahH were co-expressed,the concentration of homocysteine catalyzed by SahH could be increased from 18.6?M to 50.4?M?170.9%?.?2?Mass spectrometry results showed that phosphorylation sites only existed at position 444 in SahH.The site-directed mutant plasmid pETDuet-sahH-M444 was successfully constructed,and the catalytic activity of the mutant recombinant protein SahH was analyzed.The results showed that after mutantion,the homocysteine concentration decreased from 100.2?M to 21.8?M?78.24%?;?3?Mutation in positions 337 and 338 of the critical site of the open and closed conformation of the effect on identification,The single mutation and double mutation were performed on amino acids337 and 338 respectively,and the single mutant plasmids pETDuet-sahH-M337 and pETDuet-sahH-M338 and the double mutant pETDuet-sahH-M337-338 were successfully constructed.The mutated recombinant protein SahH was catalyzed in vitro.The results showed that after mutation,the homocysteine concentration of each mutated recombinant SahH decreased from 100.2?M to 1.6?M,13.8?M and 4.5?M,respectively.Through the development of this study,the optimal method for the detection signal molecule AI-2of V.harveyi BB170 was established and used to detect the AI-2 activity of Brucella.The results showed that there was no activity of AI-2 in Brucella,and it used sahH activated methionine cycle.Through optimization of the conditions affecting the catalytic reaction of SahH,its optimal reaction system was established;on this basis,the catalytic active sites of SahH were identified.This study will lay the foundation for further research on the function of SahH.