Study On The Mechanism Of GltB Affecting The Formation Of Persister In Staphylococcus Aureus

Staphylococcus aureus is the most common pathogen in humans and can produce a variety of virulence factors that can cause both localized septic infections of the skin or tissues,as well as infections of internal organs and even systemic threats.Persister are a small subpopulation of bacteria in the bacterial population that is insensitive to antibiotics and is essentially in a dormant state in which bacteria can evade killing by antibacterial drugs as well as the host immune response.In our laboratory,we obtained a knockout strain of glutamate synthase,a large subunit gene(gltB)in a previous study,and found that the deletion of gltB leads to reduced persister and L-form,staphyloxanthin formation ability of S.aureus.Objective:To explore the molecular mechanism of gltB affecting the formation of persister,cell wall,and staphyloxanthin synthesis of S.aureus Newman strain for providing a new drug target for the prevention and control of S.aureus persister infection.Methods:1.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of various antibacterial agents against the S.aureus Newman strain andΔgltB strain were determined by the microdilution method.2.Different antimicrobial exposure(ampicillin and levofloxacin)tests were performed to determine the persister formations of Newman strains andΔgltB strain at different time points of culture.Formation of Newman strain,ΔgltB strain,andΔgltB strain persister with exogenous glutamate supplementation were determined.3.The growth curves of WT::pRAB11,ΔgltB::pRAB11,andΔgltB::pRABgltB under anhydrotetracycline induction and resistance to heat,acid,oxidants,and starvation were determined.Exposure tests to different antimicrobial drugs(ampicillin,norfloxacin,gentamicin,and vancomycin)were performed to determine the formation status of persister in the three strains.4.The precipitates of Newman strain andΔgltB strain cultured for 48h were collected for transcriptomic sequencing(RNA-seq),followed by bioinformatics analysis.Q-PCR was used to validate the results.5.Different antimicrobial exposure tests were used to observe the formation of persister in Newman strain,ΔgltB strain,andΔgltB strain with exogenous supplementation of histidine.Q-PCR was performed to determine the expression levels of related genes in Newman strain andΔgltB strain at different time cultural points.6.The Newman strain,ΔgltB strain,and the exogenous supplemented glutamate and histidine ofΔgltB strain cultured to 18h were inoculated into an L-form induction medium and the L-form formation abilities of the four bacterial suspensions were observed.7.The cellular ultrastructures of S.aureus Newman strain andΔgltB strain were measured by transmission electron microscopy(TEM).The biomechanical properties of the two strains were examined by atomic force microscopy(AFM),and the expression levels of related genes in Newman strain andΔgltB strain were determined by Q-PCR.8.The precipitates of WT::pRAB11,ΔgltB::pRAB11 andΔgltB::pRABgltB strains were collected under anhydrotetracycline induction at 6h and 48h of incubation to compare the differences in staphyloxanthin formation and determine the levels of staphyloxanthin;Q-PCR was performed to determine the expression levels of related genes in the three strains.Results:1.Compared to the Newman strain,gltB was more sensitive to antimicrobial drugs.Its MIC and MBC to a variety of antimicrobial drugs were lower than those in the Newman strain2.In comparison to Newman strain of 48h cultures with numerous viable bacteria presenses at 8d and 6d under the action of ampicillin and levofloxacin,respectively,the ability ofΔgltB strain to form persister under the action of ampicillin and levofloxacin was significantly reduced,as shown by the absence of viable bacteria.After exogenous supplementation with glutamate,the tolerance of theΔgltB strain to ampicillin was significantly enhanced with viable bacteria at around 10~6CFU/m L,as compared withΔgltB strain at around 9d exposure to ampicillin in the absence of viable bacteria.The same results were observed in the levofloxacin essay that theΔgltB strain had no viable bacteria after 7d exposure,whileΔgltB strain with glutamate supplementation were not dead until 10d exposure.3.The growth rates of WT::pRAB11,ΔgltB::pRAB11andΔgltB::pRABgltB under anhydrotetracycline induction were slower than that of WT::pRAB11 without anhydrotetracycline induction in the earlier 16h culture.WT::pRAB11 is more resistant to heat,acid,oxidant,and hunger thanΔgltB::pRAB11.ΔgltB strains partially recovered their resistance to the above pressure after supplementing gltB.WT::pRAB11,ΔgltB::pRAB11 andΔgltB::pRABgltB of 3h cultures were sensitive to ampicillin,norfloxacin,gentamicin and vancomycin at.The tolerances of WT::pRAB11 of 24h,48h,and 72h cultures to the four antimicrobials were significantly stronger than that ofΔgltB::pRAB11.And the tolerance ofΔgltB::pRABgltB to the four antimicrobials recovered after gltB supplementation.4.RNA-seq results showed that a total of 184 Differentially Expressed Genes(DEGs)were detected in theΔgltB strain compared with the Newman strain.Eight of them were up-regulated and 176 of them were down-regulated.All the DEGs were mainly distributed in histidine metabolism,microbial metabolism,quorum sensing,ABC transport system,biosynthesis of secondary metabolites and two-component system.5.After exogenous supplementation with histidine,the tolerance of theΔgltB strain to ampicillin was significantly enhanced with viable bacteria at around10~6CFU/m L,as compared withΔgltB strain at around 9d exposure to ampicillin in the absence of viable bacteria.The same results were observed in the levofloxacin essay that theΔgltB strain had no viable bacteria after 7d exposure,whileΔgltB strain with glutamate supplementation was still have 10~3CFU/m L.Q-PCR experiments showed that the expression levels of the genes his F,his H,his C,and his D related to histidine synthesis inΔgltB strain were significantly lower than those in Newman strain at 24h,48h,and 72h of culture(P<0.05).6.Newman strain andΔgltB strain supplemented with glutamate/histidine were cultured in LIM medium for 72h,and the typical"fried egg"-like colonies were observed,while the number of"fried egg"-like colonies formed byΔgltB strain was small.7.The cell wall ofΔgltB strain was significantly thinner than that of Newman strain(76.07±8.16 nm vs 38.70±6.60 nm)(P<0.05).Under atomic force microscopy,the surface ofΔgltB was rougher and more incomplete than that of Newman,and the Young’s modulus ofΔgltB(8.60±2.20 k Pa)was significantly lower than that of Newman(14.12±3.35 k Pa).The cell wall ofΔgltB strain was softer than that of Newman strain.The expression levels of cell wall synthesis related genes NWMN＿1063,mru D,mra Y and fem B inΔgltB strain were significantly lower than those in Newman strain(P<0.05).8.The staphyloxanthin produced byΔgltB::pRAB11 was significantly less than that by WT::pRAB11 after culture for 24h,and the staphyloxanthin synthesis ability recovered after gltB supplementation(P<0.05).The expression levels of staphyloxanthin synthesis-related genes(mvas,crt Q,crt M)inΔgltB::pRAB11 were significantly lower than those in WT::pRAB11,and the expression levels of related genes recovered after gltB supplementation(P<0.05).Conclusion:1.gltB affects the formation of persister and L-form in S.aureus in the stationary late phase and is closely related to the synthesis of glutamate and histidine.2.The deletion of gltB can affect the synthesis of S.aureus cell wall,and its mechanism is related to gltB knockdown leading to reduced L-glutamate production,which in turn triggers a reduction in the expression level of NWMN＿1063.3.The deletion of gltB leads to reduced expression of relevant genes in the staphyloxanthin synthesis pathway,thereby affecting the synthesis of staphyloxanthin in S.aureus.