Research On Mechanism Of Antibiotic Tolerance Mediated By CPA In Staphylococcus Aureus

"Healthy breeding"mode is a fundamental guarantee and inevitable trend to realize the vigorous development of China's dairy industry,which points out the direction for standardizing the intensive breeding of dairy cows,improving the health of dairy cows and reducing the abuse of antibiotics.Bovine mastitis has seriously hindered the healthy development of dairy industry,and become an important factor restricting the rapid development of healthy dairy breeding mode because of its frequent occurrence and poor treatment effects.Staphylococcus aureus is one of the main pathogens of bovine mastitis.It can not only resist environmental stress by regulating its own metabolic processes,but also escape immune killing by entering host cells.So that it's very difficult to be eradicated in the process of disease infection.Although the use of antibiotics greatly improves the therapeutic effect of bovine mastitis,also induces a variety of resistant strategies to antibiotics in bacteria,which that antibiotic tolerance is one of them.In addition,there are researches show that antibiotic tolerance is not only the basis of evolution in drug resistance,but also the key to infection and recurrence of clinical chronic diseases.Cation/proton Antiporters(CPA)are a set of transport systems which catalyze active efflux of Na⁺in exchange for H⁺from outside the bacteria.It can help bacteria maintain the stability of intracellular p H and osmotic pressure in face of extreme environments.It has been reported that CPA can affect the growth and metabolism of bacteria.However,there is no report on whether CPA can lead to antibiotic tolerance phenotype in bacteria.In the early stage of this experiment,Staphylococcus aureus Newman was selected for the induction experiment in vitro and the induced strains with tolerant phenotype were obtained.The whole genome sequencing revealed a key mutated gene NWMN?0545,while the proteomic analysis revealed key genes including cpa1-1.Later,I focused my research on how CPA mediated the multiple antibiotic tolerance phenotype of Staphylococcus aureus by means of molecular biology.Results are as follows:(1)Mutated NWMN?0545 leads to multiple antibiotic tolerance phenotype in Staphylococcus aureus.Strains(Dic-T5-20,Nafc-T5-17,Fluc-T5-20 and Cefe-T5-17)with tolerance phenotype were successfully induced through induction of four antibiotics(Cefepime?Dicloxacillin?Flucloxacillin?Nafcillin)in vitro.The mutation gene NMWN?0545was identified in all four tolerant strains by whole genome sequencing.The mutation of NWMN?0545 was resulted in the appearance of tolerance phenotype by constructing the complementation strain(0545^mut-p SC1-P0545),knockout strain(Newman-?0545)and determining killing curve.Through the detection of growth phenotype and cross-tolerance phenotype,we found that 0545^mut strain not only had a prolonged lag phase(P<0.01),but also had the phenotype of multiple antibiotic tolerance for Penicillins?Cephalosporins?Carbapenems?Glycopeptides and Quinolones(P<0.01).(2)The down-regulation of cpa1-1 leads to the production of antibiotic tolerance phenotype in 0545^mut strain.Because the function of NWMN?0545 is unknown,so we sent0545^mut strain to proteomics sequencing.According to the sequencing results,the expression of Na⁺/H⁺antiport gene cpa1-1 was significantly down-regulated(P<0.01),and the expression change of cpa1-1 was verified by q PCR;The determination of extracellular p H in0545^mut strain showed that Na⁺/H⁺antiport decreased;Down-regulated expression of cpa1-1resulted in a prolonged lag phase and tolerance phenotype in 0545^mut strain by constructing the overexpression strain(0545^mut-p SE1-cpa1-1),knockout strain(Newman-?cpa1-1)and determining killing curve.(3)The decrease of Na⁺/H⁺antiport resulted in antibiotic tolerance phenotype in Staphylococcus aureus.According to the detection of killing curve in Newman wild-type under different p H and high Na⁺or K⁺concentration,we proved that Newman had tolerance phenotype and prolonged lag phase phenotype under the condition of strong acid(p H=4.5)(P<0.01);it also had tolerance phenotype under high Na⁺or K⁺concentration(1M Na Cl or KCl),but the effect on lag phase was not as significant as p H.In this experiment,we proved that the decrease of Na⁺/H⁺antiport could mediate the antibiotic tolerance phenotype through the detection of antibiotic phenotype in Newman wild-type under the condition of artificial inhibition of Na⁺or H⁺antiport function in Staphylococcus aureus.(4)Preliminary study on the correlation between cpa1-1 and NWMN?0545.Through IP test,Mgr A,Sar A,Sar Z,Pur R and other proteins binding to NWMN?0545 were fished out;Through DNA Pull-down test,Sara,Sar R,Rzc A,Agr A and other regulatory proteins binding to cpa1-1 promoter were fished out;Through mass spectrometry analysis,the related candidate regulatory genes were found out:mgr A,sar A,sar R,sar X,sar Z,rzc A,pur R,agr A.We want to explore the regulation relationship between cpa1-1 and NWMN?0545,which lead to the decreased function of Na⁺/H⁺antiport in this experimentIn conclusion,we successfully obtained 0545^mut strain with multiple antibiotic tolerance phenotype and proved NWMN?0545 and cpa1-1 were directly related to the antibiotic tolerance phenotype by gene editing technology.We proved that the decrease of Na⁺/H⁺antiport could mediate the antibiotic tolerance phenotype in Staphylococcus aureus,and explored a regulatory pathway"Antibiotics?NWMN?0545 mutation?Decrease of Na⁺/H⁺antiport(decreased expression of cpa1-1)?multiple antibiotic tolerance phenotype"by measuring the changes of extracellular p H in 0545^mut strain,and the phenotype of Newman wild-type to antibiotics under different p H and high Na⁺or K⁺concentration.The elucidation of antibiotic tolerance mechanism in Staphylococcus aureus not only provides theoretical basis for effective treatment of bovine mastitis infection,but also provides basis for reducing antibiotic abuse and promoting the development of healthy breeding mode in dairy cows.