Analysis On The Mechanism Of Persister Formation In Staphylococcus Aureus Using Transcriptomics And Metabolomics

Persister cells are a multi-drug tolerant subpopulation within an isogenic culture of bacteria,which are responsible for chronic and refractory infections.In addition,the presence of persisters can lead to the occurrance of drug-resistant mutants and are associated with prolonged antibiotic therapy.The mechanism of persister formation in Staphylococcus aureus（S.aureus）is poor knowledged and requires a deep understanding of its molecular mechanisms.Concerning the essentail role of metabolic control in persister cells,this project employs methods of metabolomics and analysis of integration of metabolomics and transcriptomics to explore the underlying metabolic pathways that may be involved in the formation of S.aureus persisters.Objective: 1.To explor metabolic pathways that may be involved in the formation of S.aureus persisters from perspective of transcriptional and metabolic levels.2.To explore the effects of amino acid metaboism on the S.aureus persister cells.Methods: 1.After the culture was 1:1000-fold diluted by TSB,S.aureus was cultured in 37°C with shaking to designated time points（including 3 hours（hrs）,4 hrs,5 hrs,6 hrs,9 hrs,and 24 hrs）.Besides determining the number of bacteria using colony forming units（CFUs）,antibiotics including ampicillin and norfloxacin exposure tests were used to measure the ratio of persisters.2.The bacteria cultured at 3 hrs,4 hrs,and 5 hrs as mentioned above were collected and employed as samples for performing untargeted metabolomics.The results were conducted to statistical and bioinformation analysis including PCA,PLS-DA,OPLSDA,univariate statistical analysis,cluster analysis,and enrichment analysis,and a set of pathways that may be responsible for S.aureus persister formation were obtainned.Metabolomics combined with transcriptomics to explore the pathways that may be involved in the bacteria persistence in S.aureus.3.QPCR were employed to detect the profiles of gene expression in alanine,aspartate and glutamate metabolism pathway and arginine biosynthesis pathway.4.Based on homologous recombination,glt B,which recodes large subunit of glutamate synthase,incolved in aspartate and glutamate metabolism pathway was knocked out using plasmid p MX10.Subsequently,S.aureus Newman wild strain and Δglt B were conducted antibiotic treatment to measurement of persister levels at designated time points covering 3 hrs,4 hrs,5 hrs,6 hrs,9 hrs and 24 hrs.Moreover,growth on TSA,hemolysis test in sheep blood agar,and coagulase test were performed to explore the characteristics of the bacteria after deletion of glt B.Results: 1.After exposure to the lethal dosage of antibiotics,the levels of persisters are inhomogeneity.There was a similar overall trend of increased persister cells with prolonged incubation.The bacteria in 3 hrs and 4 hrs’ cultures could be eradicated by antibiotics efficiently,while it became extremely difficult to be eradicated after or more than 5 hrs incubation by antimicrobial agents.2.With the standardization of p<0.05 and variable importance for the projection（VIP）≥ 1,there were 42 significantly changed metabolites comparing 4 hrs with 3 hrs samples,including 39 accumulated metabolites and 3 consumed metabolites.The changed metabolites are mainly enriched in nucleotide metabolism,membrane transport,amino acid metabolism,and genetic information processing pathways.When comparing 5 hrs with 3 hrs samples,there were 45 accumulated metabolites and 3 assumed metabolites identified.After enrichment analysis,these changed metabolites are enriched in the pathways of membrane transport,nucleotide metabolism,lipids metabolism,vitamin and cofactor metabolism,amino acid metabolism,biosynthesis of secondary metabolites,and genetic information processing pathways.There had been identified 59 metabolites comparing 5 hrs with 4 hrs samples,which includes 49 accumulated metabolites and 10 consumed metabolites.These changed metabolites cover a wide range of pathways,mainly enriched in membrane transport,vitamin and cofactor metabolism,nucleotide metabolism,lipid metabolism,carbon metabolism,amino acid metabolism,biosynthesis of secondary metabolites,and genetic information processing pathways.3.Combined the results of transcriptomics and metabolomics,a set of metabolic pathways that may be involved in S.aureus persister formation,including carbohydrate metabolism（mainly referring to pyruvate metabolism,glycolysis / gluconeogenesis,and tricarboxylic acid cycle）,amino acid metabolism（mainly referring to amino acid biosynthesis）,cofactors and vitamins metanolism（mainly referring to nicotinate and nicotinamide metabolism）,and nucleotide metabolism pathways were identified.4.After detection of gene expression profiles with q PCR,the expression leve of genes involved in arginine metabolic pathway and glutamate and aspartate metabolic pathway were examined.gud B,glt B,glt D,NWMN₂454,gln A,and NWMN₂065 were up-regulated in 5 hrs（p<0.05）.On contrary,NWMN₀126,arg G,arg H,arg F,arc B,arc A,arg J,arg C,pur B,pur A,and pyr B were down-regulated at the same time point.5.After construction of Δglt B,ampicillin and norfloxacin were employed to explore the ratio of persister cells in S.aureus wide type and the mutant.The results showed that the persister levels are identical in wide type and Δglt B as cultured to 3 hrs,4hrs,5 hrs,6 hrs but inhomogeneity in bacteria cultured to 9 hrs and 24 hrs,and displayed that the mutant could be eradicated more easily by the antibiotics than the wild type.6.Δglt B appears to reduce production of pigment and coagulase,but promote hemolysis.Conclusion: 1.There was an overall trend of increased persister cells with prolonged incubation,and the persister will formed boomingly at a certain phase.A list of metabolic pathways including carbohydrate metabolism（mainly referring to pyruvate metabolism,glycolysis / gluconeogenesis,and tricarboxylic acid cycle）,amino acid metabolism（mainly referring to amino acid biosynthesis）,cofactors and vitamins metanolism（mainly referring to nicotinate and nicotinamide metabolism）,and nucleotide metabolism pathways responsible for S.aureus persister formation.2.Arginine metabolic pathway and glutamate and aspartate metabolic pathway are accountable for forming persister cells in S.aureus.glt B palys a criticl role in enhancing the bacteria to maintain persistence.