Transcriptome-Metabolome Combined To Explore The Mechanism Of PurN Affecting The Formation Of Persister And Virulence In Staphylococcus Aureus

Objective:To explore the molecular mechanism of purN affecting the formation of persister and virulence in Staphylococcus Newman strain,and to find new drug targets for the prevention and treatment of S.aureus persister infection.Methods:1.The overnight cultured S.aureus Newman strain and ΔpurN were diluted 1:1000 by TSB and cultured for 3h,4h,5h,9h,and 24h,respectively.Ampicillin（10 μg/mL）exposure experiments were then performed with the cultures to observe the status of persister formation.2.The recombinant plasmids pRAB11-purN were extracted from the previously constructed complementing strain ΔpurN::pRBpurN,and verified by double digestion with restriction endonuclease and sequencing.The pRAB11 and pRAB11-purN were electroporated into S.aureus Newman strain（WT）and ΔgltB competent cells,respectively,to obtain purN overexpressing strains WT::pRBpurN andΔgltB::pRBpurN as well as control strain ΔgltB::pRAB11.The above bacteria were collected after they were induced in TSB containing 100 ng/mL anhydrotetracycline（ATc）for 5h,and the expression level of purN was determined by RT-qPCR.3.The growth curve of the constructed strains of S.aureus cultured in TSB contained ATc were determined.Ampicillin（10μg/mL）,levofloxacin（20μg/mL）,vancomycin（40μg/mL）and gentamicin（100μg/mL）exposure experiments were used to determine the persister forming ability of WT::pRAB 11,ΔpurN::pRAB11,ΔpurN::pRBpurN and WT::pRBpurN which were cultured for 5 h,9 h and 18 h,respectively.4.The bacteria of Newman strain（NWE）and ΔpurN（PurNE）which cultured for 5h were diluted 1:1000,and the corresponding sampled which were treated with ampicillin for 3h（NWA and PurNA）were collected for transcriptome and non-targeted metabolome detection.And a combined transcription-metabolism analysis was performed.The transcriptome results were validated by RT-qPCR.The protein-protein interaction network analysis diagram of differential genes was constructed by STRING software to analyze the key genes affecting the formation and virulence of the persister.5.Ampicillin,levofloxacin,vancomycin and gentamicin exposure experiments were used to determine the persister forming ability of WT::pRAB11,ΔgltB::pRAB11,ΔgltB::pRBpurN and WT::pRBpurN after they were cultured in TSB containing ATc for 5h,9h,and 18h,respectively,to detect the persistence of different strains.RT-qPCR was used to detect the expression levels of gltB in WT::pRAB11,ΔpurN::pRAB11,ΔpurN::pRBpurN and WT::pRBpurN,and the relationship between gltB and purN expression was analyzed.6.The expression levels of saeS,saeR and main virulence genes including hla,hlgA,hlgB,hlgC,lukF,lukS,NWMN₁873,NWMN 1926,eta and sea in WT::pRAB11,ΔpurN::pRAB11,ΔpurN::pRBpurN and WT::pRBpurN were detected by RT-qPCR;The hemolysis and biofilms formation ability of the four strains were observed.Result:1.The results of the ampicillin exposure experiment showed that the Newman strain and ΔpurN had the significant difference in the persister forming ability when cultured for 5h.However,after culturing for 9h and extension,the difference disappeared.2.The sequence of the recombinant pRBpurN was validated that had no base mutation and was suitable for further research.The purN gene overexpression strains WT::pRBpurN and ΔgltB::pRBpurN were constructed successfully.The purN gene expression level in WT::pRBpurN and ΔgltB::pRBpurN was significantly higher than that in WT::pRAB11 after they were cultured in TSB with ATc（P<0.05）.3.The growth of different strains in TSB containing ATc was inhibited to a certain extent,and the time to reach the stationary phase was delayed.The cultures of WT::pRAB11,ΔpurN::pRAB 11,ΔpurN::pRBpurN（complemented strain）and WT::pRBpurN（overexpressing strain）at 5h,9h and 18h showed differences in the persister forming ability against different antibiotics.When cultured for 5h,the four strains were all highly sensitive to different antibiotics.However,when cultured for 9h,all the strains were killed after exposed to antibiotics 3 days,except for WT::pRBpurN which exhibited higher ability of persister formation than other strains and was all killed after 5 d of exposure.After culturing for 18 h,these difference disappeared.4.The metabolomics test discovered that when PurNE compared with NWE,there were 70 kinds of differential metabolites detected totally,of which 22 were up-regulated and 48 were down-regulated;when PurNA compared with NWA,there were 68 kinds of differential metabolites detected totally,of which 23 were up-regulated and 45 were down-regulated;when NWA compared with NWE,there were 119 kinds of differential metabolites detected totally,of which 47 were up-regulated and 72 were down-regulated;when PurNA compared with PurNE,there were 130 kinds of differential metabolites detected totally,of which 54 were up-regulated and 76 were down-regulated.These differential metabolites were mainly enriched in amino acid biosynthesis and metabolism,ABC transporters,energy metabolism,nucleotide metabolism and other pathways.5.Transcriptome sequencing discovered that when PurNE compared with NWE,there were 88 kinds of differentially expressed genes（DEGs）screened totally,of which 25 were up-regulated and 63 were down-regulated;when PurNA compared with NWA,there were 69 kinds of differentially expressed genes（DEGs）screened totally,of which 3 were up-regulated and 66 were down-regulated;when NWA compared with NWE,there were 127 kinds of differentially expressed genes（DEGs）screened totally,of which 100 were up-regulated and 27 were down-regulated;when PurNA compared with PurNE,there were 116 kinds of differentially expressed genes（DEGs）screened totally,of which 64 were up-regulated and 52 were down-regulated.The main enriched pathways of down-regulated DEGs were mainly related to S.aureus infection,amino acid biosynthesis and metabolism,secondary metabolite biosynthesis and twocomponent system.The up-regulated DEGs were mainly related to ABC transporters,aminoacyl-tRNA biosynthesis,signal transduction and other pathways.6.Transcription-metabolism combined analysis showed that DEGs and differential metabolites are mainly enriched in amino acid biosynthesis and metabolism,secondary metabolite biosynthesis,purine and pyrimidine metabolism,aminoacyl-tRNA biosynthesis,carbon metabolism and other pathways.Protein-protein interaction network analysis revealed a correlation between the expression of gltB,saeS,saeR and purN.7.RT-qPCR detection showed that with the higher expression level of purN,the more expression of gltB in WT::pRBpurN and ΔpurN::pRBpurN than that in wild strain（P<0.05）.The number of viable bacteria in ΔgltB::pRAB11 and ΔgltB::pRBpurN were lower than those in WT::pRAB11 and WT::pRBpurN when cultured to different time（5h,9h,18h）and exposed with antibiotics,and the persister forming ability of WT::pRBpurN was higher than that of ΔgltB::pRBpurN.8.With the difference in the expression of the gene purN,saeS and saeR in WT::pRAB11,ΔpurN::pRAB11,ΔpurN::pRBpurN and WT::pRBpurN showed similar changes to purN.At the same time,the time（10h）of the hemolytic ring appeared on blood TSA contained with ATc of WT::pRBpurN was earlier than that of other strains（14h）,and the hemolysis ability of ΔpurN::pRAB 11 was significantly decreased.The hemolytic ability of WT::pRAB11 and ΔpurN::pRBpurN tended to be consistent,and the hemolytic ability of ΔpurN was restored after purN was complemented.The biofilms forming ability of WT::pRAB11,ΔpurN::pRBpurN and WT::pRBpurN was significantly stronger than that of ΔpurN::pRAB11（P<0.05）.9.The expression levels of major virulence genes including hla,hlgA,hlgB,hlgC,lukF,lukS,NWMN₁873,NWMN₁926,eta,sea and coa in WT::pRAB11 were significantly higher than those in ΔpurN::pRAB11（P<0.05）.The expression levels of most virulence genes were restored in ΔpurN::pRBpurN.After overexpression of purN in WT::pRBpurN,the expression levels of lukS,lukF,hla and coa were significantly higher than those in WT::pRAB11（P<0.05）.Conclusion:1.By affecting the expression of gltB in S.aureus,purN regulates in the synthesis and metabolism of glutamate,and participates in the formation of persister in S.aureus at late-exponential phase.At the same time,energy metabolism,ABC transporters,biosynthesis of others secondary metabolites,purine and pyrimidine metabolism,carbon metabolism and other pathways are also involved in the roles of purN in formation of persister.2.purN affects the expression of virulence factors and formation of biofilms in S.aureus through the two-component system saeRS,thereby affecting the bacterial virulence.