Analysis On The Mechanism Of Staphylococcus Aureus Unstable L-form Formation Based On Metabolomics

Bacterial L-form refers to the cell wall-deficient bacteria,which can be formed in vivo,in vitro and under artificial induction,and is resistant to antibiotics acting on the cell wall.Staphylococcus aureus can form L-form,which is closely related to chronic and relapsing infection.At present,the mechanism of S.aureus L-form formation is unclear.This project aimed to explore the mechanism of S.aureus L-form formation from the metabolic level.Objective:1.Based on the results of metabolomics of L-form of S.aureus Newman strain,the metabolic changes during the formation of L-form were discussed.2.To explore the effects of glt B on the formation of S.aureus L-form,persisters,biofilms and its virulence.Methods:1.The L-form induction medium（LIM）was prepared and inoculated with S.aureus Newman strain in stationary phase in order to induce and establish the model of S.aureus unstable L-form.2.The S.aureus unstable L-form induced and cultured on LIM for 72 hours and its normal-form（N-form）cultured on control medium were collected by PBS repeated washing.The samples were detected by untargeted metabolomics and the different metabolites were analyzed by bioinformatics.The metabolic characteristics of S.aureus L-form were explored,and the pathways related to the formation of S.aureus L-form were screened.3.The wild strain of S.aureus and glt B mutant in stationary phase were inoculated with LIM and induced.The abilities of L-form formation in wild strain andΔglt B were observed.4.glt B of S.aureus Newman strain was amplified by PCR.After the products were digested by Eco R I and Bgl II,the fragments were ligated with plasmid pRAB11 and transformed into Escherichia coli DC10B.Then the recombinant plasmid pRAB11-glt B was obtained.The pRAB11 and pRAB11-glt B were electroporated into wild strain andΔglt B of S.aureus respectively,the WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B were screened.Total RNA of WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B which were cultured in TSB containing 100ng/m L anhydrotetracycline（Atc）was collected and their glt B expression levels were determined by q-PCR.The three strains were inoculated on TSA plate containing Atc for 24 hours,and the growth characteristics and pigment production abilities of each strain were observed.5.WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B in stationary phase were inoculated on LIM containing Atc respectively,and then were induced to form bacterial L-form.The ability of the L-form formation and the growth of the three strains were observed continuously.6.Antibiotic exposure assay was used to determine the sensitivities of WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B to ampicillin（20μg/m L）and norfloxacin（20μg/m L）cultured 3h,4h,5h,6h,9h and 24h in TSB containing Atc.7.WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B were inoculated in TSB containing Atc on 96 well plate.Following 24h incubation at 37℃,the planktonic bacteria were rinsed away,and the biofilms were stained with crystal violet.After observed and pictured,the stained biofilms were solubilized in 33%acetic acid and the optical density（OD）of the solution was measured by spectrophotometry with a wavelength of 570 nm.8.LD₅₀ of the wild strain andΔglt B of S.aureus to female BALB/C mice were used to determine the effect of glt B knockout on bacterial virulence by intraperitoneal injection.The expression levels of the main virulence genes including hla,hlg A,hlg B,hlg C,luk D,luk E,luk F,luk S,NWMN＿1503,NWMN＿1873,NWMN＿1926,NWMN＿2071,eta,and sea of the wild strain andΔglt B of S.aureus were detected by q-PCR.Results:1.S.aureus Newman strain formed unstable L-form after 72 hours of induction on LIM,and the colonies were presented as typically like“fried eggs”under the inverted microscope.2.The results of the untargeted metabolomics assay showed that there were 138different metabolites in S.aureus L-form cultured for 72 hours compared with N-form,of which 62 were increased and 76 were decreased,indicating that the overall metabolism of S.aureus L-form was slightly active.Differential metabolites were mainly enriched in ABC transport system,amino acid biosynthesis and metabolism,nucleotide metabolism,two-component system,carbon metabolism,nitrogen metabolism,oxidative phosphorylation,glutathione metabolism and other pathways.3.Compared with the wild strain of S.aureus,the ability ofΔglt B L-form formation decreased significantly.The colonies ofΔglt B L-form at 72h were small with atypical morphological characteristics,and the quantity of colonies is poor.Some of them were like“fried eggs”,and their growth speed of colonies was significantly slower than that of the wild strain.4.In this study,WT::pRAB11,Δglt B::pRAB11 andΔglt B::pRABglt B were successfully constructed.The glt B gene was highly expressed inΔglt B::pRABglt B after induced by Atc.All the three strains could form S-type colonies on TSA containing Atc,but WT::pRAB11 had a significantly stronger ability to produce golden yellow pigment thanΔglt B::pRAB11 and the complemented strain.5.WT::pRAB11 and the complemented strain could form typically“fried eggs”liked colonies after 72 hours of induction on LIM,and the number of colonies of the complemented strain was slightly less than that of WT::pRAB11.A small amount of“fried eggs”liked colonies were formed inΔglt B::pRAB11,and the volume of colonies was small with a small amount of"granular"colonies could be seen.These results indicated that the ability of the L-form formation ofΔglt B::pRAB11 was significantly lower than that of WT::pRAB11 and complemented strain.6.There was no significant difference in the sensitivities of WT::pRAB11,Δglt B::pRAB11 and complemented strain to ampicillin and norfloxacin cultured for3h,4h,5h and 6h in TSB containing Atc.However,after 9h cultivation,Δglt B::pRAB11 was killed after exposure to ampicillin and norfloxacin for 5d and 9d respectively,while WT::pRAB11 was killed after exposure for 7d and 10d respectively.After 24h cultivation,Δglt B::pRAB11 was killed after exposure to ampicillin and norfloxacin for 11d and 7d respectively,while WT::pRAB11 still had a large number of bacteria survival after 12 days.The results showed that the ability of persisters formation ofΔglt B::pRAB11 was significantly lower than that of WT::pRAB11,and the complemented strain recovered the ability of persisters formation of ampicillin and norfloxacin.7.After 24h of culture in TSB containing Atc,the abilities of biofilms formation of WT::pRAB11 and complemented strain were significantly stronger than that ofΔglt B::pRAB11.The OD_570nm of WT::pRAB11 and complemented strain were 3.784±0.0173 and 3.700±0.0164 respectively,which were significantly higher than that ofΔglt B::pRAB11（2.460±0.1055,P<0.05）.The results showed that the ability of biofilms formation ofΔglt B::pRAB11 was significantly lower than that of WT::pRAB11.The ability of biofilms formation was restored after glt B was complemented.8.The LD₅₀ of wild strain to mice was 2.6×10⁹ CFU/m L,and that ofΔglt B was1.065×10¹⁰ CFU/m L,which was about 4.1 times of that of wild strain,indicating that that the virulence of the mutant decreased significantly.The expression of virulence genes in wild strain andΔglt B was determined by q-PCR.The results showed that except for hlg B,the expression levels of other 13 virulence genes including hla,hlg A,hlg B,hlg C,luk D,luk E,luk F,luk S,NWMN＿1503,NWMN＿1873,NWMN＿1926,NWMN＿2071,eta,and sea ofΔglt B were significantly down regulated than that of wild strain of S.aureus（P<0.05）.Conclusion:1.Compared with S.aureus N-form in the same period,the metabolites and metabolic pathways of S.aureus L-form are significantly changed,and the adjustment of metabolic level plays an important role in the formation and growth of S.aureus L-form.Amino acid metabolism and biosynthesis（including alanine,aspartate and glutamate metabolism,arginine biosynthesis,D-glutamine and D-glutamate metabolism etc.）,carbon metabolism,ABC transport system,nitrogen metabolism and other metabolic pathways are closely related to the formation of S.aureus L-form.2.glt B,coding the large subunit of glutamate synthase,plays an important role in the formation of S.aureus L-form,persisters and biofilms,and regulates the virulence of bacteria by influencing the main virulence factors expression.