Study On The Effects Of PurN On Persister Formation And Virulence Of Staphylococcus Aureus

Bacterial persisters are phenotype variants with high tolerance to lethal dose antibiotics and stresses,and respond for persistence and chronic infections.Staphylococcus aureus is one of the dominant pathogen in community-acquired and hospital-acquired infections.The emergence of bacterial resistance to antibiotics and persisters formation bring extremely difficulty for the treatment with S.aureus related infections.At present,the mechanisms of persisters formation in S.aureus is still unclear,and need further exploration.Based on the discovery of RNA-seq,this sdudy explores the effects of purN on the persiter formation and virulence in S.aureus.Objective:1.To explore the mechanisms of persister formation and the effects of purN on persister formation in S.aureus.2.To study the effects of purN on the virulence in S.aureus.Methods:1.S.aureus Newman strain was cultured for 3 h,4 h,5 h,9 h,24 h and then exposed to 10μg/ml ampicillin,respectively.CFU counts were determined everyday to observe persister formation at different time point.2.The total RNA of S.aureus Newman strain which was cultured for 3 h,4 h,5h were extracted.RNA-sequencing（RNA-seq）was used to detect the changes of genomic expression of S.aureus with the extension of culture time.Q-PCR was used to verify the results of RNA-seq.3.Homologous recombination was used to construct S.aureus Newman knockout strainsΔpurN,ΔnarH,andΔadh1.4.ΔpurN,ΔnarH,Δadh1 were cultured to log phase（3 h,5 h,9 h）and stationary phase（18 h）,and then exposed to 10μg/ml ampicillin,20μg/ml levofloxacin,40μg/ml vancomycin,and 100μg/ml gentamicin,respectively.CFU counts which were determined on TSA plates were used to observe the changes of persister formation.5.The strains of WT::pRAB11,?purN::pRAB11,?purN::pRBpurN of S.aureus were construced.The persister formation of wild strains,knockout strains and complementary strains was observed by drug exposure in 10μg/ml ampicillin.6.hemolysis test,coagulase test,mannitol fermentation test and LD50measurement were used to observe the effects of purN knockout on the virulence of S.aureus.7.Q-PCR was used to detect the expression of virulence genes in S.aureus wild type andΔpurN.Results:1.Exposured to lethal concentration of ampicillin,S.aureus Newman strain was highly sensitive to antibiotics when cultured for 3 h and 4 h,and all bacteria were killed after exposure to lethal concentration of ampicillin for 3 days.Significant drug tolerance occurred when S.aureus was cultured for 5 h,and all bacteria were killed untill 7 days of drug exposure.Deep tolerance to drugs formed when S.aureus was cultured for 9 h and 24 h,and a large number of bacteria still survived after exposure to lethal concentration of ampicillin for 10 days.2.RNA-seq showed that,there were 100 differentially expressed genes in S.aureus Newman strains cultured for 4 h compared with 3 h,and 70 of which were up-regulated and 30 were down-regulated.113 differentially expressed genes were detected in cultures of 5 h versus 4 h,and 29 differentially expressed genes were up-regulated and 84 were down-regulated,respectively.The total number of differentially expressed genes was 363 in the cultures 5 h compared with 3 h,and 182of which were up-regulated and 181 were down-regulated.Compared with the differentially expressed genes when bacteria were cultured from 3 h to 5 h,the continuously up-regulated genes were adh1,ald,ilvA,narG,narH,narJ,nrdD,NWMN₂288;continuously down-regulated genes were argG,lrgA,NWMN₁266,NWMN₂577,rplK,rplL,rpmE,rpsD,rpsI,rpsK,rpsP.These genes were enriched in pathways include metabolism pathways,biosynthesis pathways,ribosome,signal transduction pathways and membrane transport pathways.3.Compared with the wild type strain,S.aureus knockout strainΔpurN was exposed to lethal concentration of ampicillin,levofloxacin,vancomycin,and gentamicin,and formation of persisters were reduced at 3 h of culture,and persister formation significantly reduced when bacteria were cultured for 5 h.During the culture period of 5 h to 9 h,the tolerance of bacteria to the drug increased gradually,and there was no significant difference from the wild strain after 9 h.4.Exposured in lethal concentration of ampicillin and levofloxacin,there was no difference of susceptibility to antibiotics inΔnarH andΔadh1 compared with wild type when cultured at 3 h,5 h,9 h,18 h.5.Tolerance to ampicillin was recovered when purN complemented S.aureusΔpurN was cultured and induced in the dehydrated tetracycline for 5 h.6.After knockout of purN,the ability of hemolysis,to produce coagulase and fermentation of mannitol of S.aureus were weakened obviously.The lethal dose 50was increased from 2.81×10⁹ CFU/mL to 3.28×10¹⁰ CFU/mL.7.The expression of virulence genes including hla,hlgA,hlgB,hlgC,lukS,and lukF which are related to virulence factors hemolysin and leukocidin in S.aureusΔpurN were significantly down-regulated than the wild type（P<0.05）.There was no significant difference of the expression of NWMN₁873,NWMN₁926,NWMN₂071,lukE,lukD,NWMN₁503,eta,coa,and sea betweenΔpurN and wild type strain（P>0.05）.Conclusions:1.Persister formation in S.aureus was closely related to the cultivation time,and the number of persister cells was less when bacteria were cultured for 3 h and 4 h,while persister formation were abundant when they were cultured more than 5 h.The mechansims of persister formation related to bacterial metabolism,synthesis,ribosome,signal transduction and membrane transport pathways etc.2.purN plays important roles in regulating the persister formation of S.aureus before the bacteria were cultured to 9 h.3.purN involved in the regulation of S.aureus virulence,which is achieved by acting on the expression of hemolysin and leukocidin.