Study On The Mechanisms Of NWMN₀037 Regulating In Persister Formation And Virulence Of Staphylococcus Aureus Based On Metabonomics

Persister is a random subset of bacterial group with high tolerance to antibiotics and external pressures.In recent years,more and more studies have confirmed that persisters are the main cause of chronic persistent infections.Staphylococcus aureus is the most common pathogen causing clinical suppurative infections.The generation of antibiotics resistance and the persister formation bring great difficulties to clinical treatment.At present,the formation mechanisms of S.aureus persister has not been clarified and needs to be studied urgently.Previous studies in our group found that S.aureus NWMN₀037 regulated in the persister formation of logarithmic phase S.aureus and virulence.In this paper,the regulated mechanisms of NWMN₀037 was studied by metabonomics.Objective:Based on non-targeted metabonomics,the mechanisms that NWMN₀037 regulates persister formation of logarithmic phase S.aureus Newman strain and it's virulence are discussed.This work will provide a new insight into S.aureus persister formation and novel target for clinical treatment of S.aureus infection.Methods:1.Levofloxacin?20?g/ml?,Ampicillin?20?g/ml?and Linezolid?40?g/ml?in Tryptone Soy Broth?TSB?were chosen to expose experiments to S.aureus Newman strain?wild strain?,NWMN₀037 mutant strain and complemented strain respectively to determine the persister formation and the sensitivity of antibiotics at different growing period.2.Wild strain and NWMN₀037 mutant strain of cultured for 6 hours and 9 hours respectively were analyzed by liquid chromatography-mass spectrometry.Cluster analysis and KEGG database comparison were used to analysis the differential metabolites between the mutant strain and wild strain of cultivation for 6 hours and cultivation for 9 hoursand explore the metabolic pathway of NWMN₀037 in regulating S.aureus Newman strain persister formation.3.The total RNA of S.aureus wild strain,NWMN₀037 mutant strain and complemented strain of cultured for 6 hours,9 hours and 18 hours were extracted respectively.The expression of52 genes?such as tpi,eno,pykA,mqo,sdhA,etc.?involved in Glycolysis/Gluconeogenesis and tricarboxylic acid cycle?TCA?metabolic pathway was detected by qPCR.4.The total proteins of S.aureus wild strain,NWMN₀037 mutant strain and complemented strain of cultured for 6 hours,7 hours,8 hours,9 hours and 18 hours respectively were extracted by low-temperature ultrasound pulverization method.After protein quantification by BCA method,the concentrations of LDH in bacteria were detected by enzyme-linked immunoassay.5.The gene expression of virulence-related genes in S.aureus wild strain,NWMN₀037mutant strain and complemented strain cultured for 6 hours was detected by qPCR.And to observe the difference of plasma coagulase test.Result:1.When cultured for 6 hours,all the mutants were killed on the 3^rd day after adding Levofloxacin,on the 2^nd day after adding Ampicillin,and on the 7^th day after adding Linezolid.On9^th day after adding Levofloxacin,Ampicillin and Linezolid,there were still 1.3×10² cfu/ml,5.0×10⁵ cfu/ml and 5.2×10⁵ cfu/ml wild strains survived respectively.All the complemented strains were killed on the 2^nd day after adding Levofloxacin and on the 9^th day after adding Ampicillin.On the 9^th day after adding Linezolid,there were still 5.0×10² cfu/ml the complemented strains were survived.When cultured for 18 hours,the three strains showed similar tolerance to the three antimicrobials,and all the bacteria were killed on the 9^th day after adding Levofloxacin,and a large number of bacteria still survived on the 9^th day after adding Ampicillin and Linezolid.2.When cultured for 6 hours,there were 77 different metabolites between mutant strain and wild strain,among which 6 metabolites increased and 71 metabolites decreased.When cultured for 9 hours,there were 71 different metabolites,among which 15 metabolites increased and 56metabolites decreased.For mutant strain,there were 56 different metabolites between cultured for6 hours and 9 hours,among which 33 metabolites increased and 23 metabolites decreased.For wild strain,there were 81 different metabolites between cultivation of 6 hours and 9 hours,among which 12 metabolites increased and 69 metabolites decreased.Metabolites cluster and metabolic pathway analysis showed that the above differential metabolites were mainly distributed in Carbohydrate metabolism,Amino acid metabolism,Lipid metabolism,Energy metabolism,Nucleic acid metabolism,Environmental metabolism,Vitamin metabolism and other metabolic pathways.3.The results of qPCR detection of genes related to carbohydrate metabolic pathway showed that the expression of gpmA,gapA,ldh,ldh1,acs,pflB and pycA were up-regulated in mutant strain compared with wild strain of cultured of 6 hours.The expression of gapA,ldh,ldh1 and pflB were up-regulated,while the expression of pycA and acs were down-regulated in mutant strain compared with complemented strain of cultured of 6 hours.The expression of ldh was up-regulated,while the expression of gpmA,pdhC,pgk,porB,NWMN₂504 and sucD were down-regulated in mutant strain compared with wild strain of cultured of 9 hours.The expression of pgk and ldh was up-regulated,while the expression of gpmA,pdhC,porB,NWMN₂504 and sucD was down-regulated in mutant strain compared with complemented strain of cultured of 9hours.The expression of gpmA and citC were up-regulated,while the expression of gapB,pdhC,NWMN₀367 and sdhA were down-regulated in mutant strain compared with wild strain of cultured of 18 hours.The expression of gpmA,pdhC,citC and sdhA were up-regulated,while the expression of gapB and NWMN₀367 were down-regulated in mutant strain compared with complemented strain of cultured of 18 hours.4.The detection of LDH concentration determination showed that the mutant strain was significantly lower than the wild strain of culture of 6 hours?P<0.05?,but no significant change was observed of cultured of 7,8,9 and 18 hours.Compared with the complemented strain,the LDH concentration in the mutant strain decreased significantly in cultivation of 7 hours?P<0.05?,but there were no significant difference among culture of 6,8,9 and 18 hours respectively.Compared with wild strain,the LDH concentrations in the complemented strain decreased significantly in cultured of 6 hours,but increased significantly in cultured of 7 hours,and there were no significant difference among cultured of 8,9 and 18 hours respectively.5.qPCR detection showed that compared with wild strain,the virulence-related gene expression including NWMN₁873,NWMN₁926,hlgA,hlgB,lukF,lukS,sea,coa,eta,hla,NWMN₁503,hlgC and lukD in mutant strain were down-regulated significantly?P<0.05?.The expression of enterotoxin-related gene NWMN₁503,hemolysin-related gene hlgC and leukocidin-related gene lukD were restored to the wild strain after the NWMN₀037 was complemented.The results of coagulase test showed that more agglutinators were formed in wild strain and complemented strain than in mutant strain.Conclusions:1.NWMN₀037 affects the formation of logarithmic phase S.aureus Newman strain persister formation by regulating glycolysis/gluconeogenesis,TCA and other metabolic pathways.2.NWMN₀037 affects the virulence of S.aureus Newman strain by regulating the expression of virulence factors including hemolysin,leukocidin,enterotoxin and coagulase.