Study On Mechanisms OF Persister And L-form Formation In Staphylococcus Aureus

Staphylococcus aureus is a prominent human pathogen. Infections with S. aureus constitute a major risk to human health due to its resistance and persistence to antibiotics. Persister formation is an important cause of persistance to antibiotics and increased tolerance to external stress. S. aureus can be induced into L-form in vitro or in vivo after they lose cell wall, and become resistant to β-lactam antibiotics, and easy to form persisters simultaneously. However, the molecular mechanisms of persister and L-form bacteria formation in S. aureus are unclear.Objective:To explore the molecular mechanisms of persisters and L-form bacteria formation in S. aureus by screening Newman strain mutant library and performing further studies and analysis.Methods:1. S. aureus Newman mutant library was screened for ampicillin hypersensitivity. The NWMN0037mutant which was deficient in persister formation was identified by inverse PCR and DNA sequencing. The mutant was complemented by plasmid pT181containing NWMN0037.2. The alteration in persister formation in different periods cultures of Newman wild type, NWMN₀037mutant and its complemented strains were compared by using ampicillin （10μg/ml） and norfloxacin （20μg/ml） exposure assay.58℃water bath,100mM H2O2, starvation were used to detect changes in stress tolerance of the three strains. The inoculated plates were incubated at33℃,37℃and42℃in order to observe temperature influence after NWMN0037was mutated.3. Crystal violet staining, OD590reading and scanning electron microscopy observation were used to detect changes in bacterial biofilm formation in NWMN0037mutant. The survival of bacterial strains in THP1macrophage-like cell were counted after they were swallowed2,4,8,24hours in order to analyze the changes in ability to survive. BALB/c mice were used to determine LD50against Newman wild type, NWMN₀037mutant and its complementation strains. Subcutaneous injection with different strains was performed and the number of viable cells in the lesions was counted after they were infected after48hours. The pathological changes were observed under the microscope.4. Real-time PCR was used to analyze the expression changes of NWMN₀037in Newman strain during the culture. Transcriptome sequencing （RNA-seq） was used to analyze genome-wide transcriptional changes after the wild strain and NWMN₀037mutant were cultured6hours and12hours （3hours after norfloxacin addition）. Hemolysis, mannitol decomposition and pigment formation tests were used to verify the results of RNA-seq. NWMN₀037was analyzed for its possible role in persister formation and virulence.5. S. aureus L-form induction medium （LIM） was repaired and culture conditions optimized. Bacterial L-forms were confirmed by observing "fried eggs" colonies, watching the morphology under electron microscope and Gram’s staining. Newman mutants library were screened by using LIM. The mutants which were deficient in L-form formation were identified by following inverse PCR, DNA sequence, and alignment. Real-time RT-PCR was used to assess the level of expression of the8mutated genes transcription in S. aureus L-form versus classical form bacteria. Complementation of glpF mutant was performed by transferred into pT181inserted with glpF. Analyze the roles of glycerol metabolism-related genes and other mutations in S. aureus L-form formation.Results:1. We obtained1mutant which was deficient in persister formation after ampicillin exposure. The mutated gene was identified to be NWMN₀037. The mutant was complemented successfully by using pT181containing NWMN₀037.2. Bacterial count results showed that after the late log phase cultures were exposed to ampicillin and norfloxacin7days, NWMN₀037mutant all died, while the wild type and the complemented strains still had viable cell numbers （logio CFU） were7.71±0.24and7.35±0.06,7.95±0.16and7.10±0.18, respectively. The difference in persistence to ampicillin and norfloxacin was lost among the3strains in early log phase and stationary phase cultures. The NWMN₀037mutant had a lower persistence to extracellular stresses than the wild type strain, and the defect was restored after complementation. The mutant strain grew more slowly at33℃than wild type and complemented strains, but the difference was lost at37℃and42℃. 3. The ability to form biofilm of the NWMN₀037mutant was decreased compared with the wild type strain, and this defect could be restored after complementation. The optical density of the mutants （0.208±0.053） was significantly lower than the wild type strain （1.025±0.493） and complemented strain （1.057±0.188）（P<0.05）. The mutant viable bacterial number （log10CFU） in THP1cells （3.30±0.03） was lower than the wild type strain （4.84±0.20） and complemented strain （4.13±0.04） at24hours. The LD50of mutant was above8.33×1010CFU/ml, while the wild type and complemented strains were2.41×109CFU/ml and1.21×1010CFU/ml, respectively. The pathogenicity of the mutant as measured by tissue damage was more severely weakened than the wild type and complemented strain. The mutant viable cell number（log10CFU） in the lesions （3.09±0.12） was less than the wild strain （4.51±0.45） and complemented strain （4.02±0.10）48hours after injection.4. During the period of5-12hours when the Newman strain was cultured in TSB medium, the expression of NWMN₀037was downregulated. Compared with wild and mutant strains at6hours culture, there were135genes upregulated and these genes distributed in several pathways, including transport, metabolism, virulence, immune evasion, regulation, stress response, cell wall and capsule biogenesis, and miscellaneous. There were466genes downregulated and most of them impacted metabolic pathways. At12hours culture （after norfloxacin addition for3hours）, there were42genes upregulated and these genes distributed in pathways of transport, metabolism, virulence （Cytotoxin associated genes hla, hlgA, hlgB, hlgC, lukF, lukS and cellular proteases associated genes NWMN₁067, chp, ssp）, immune evasion, regulation, stress response, cell division and miscellaneous. There were611genes downregulated and most of them also impacted metabolic pathways. There were39genes upregulated in the wild type strain before and after norfloxacin treatment, while these genes were not upregulated in the mutant. These genes were distributed in the pathways of metabolism, immune evasion, regulation, and miscellaneous. Hemolysis, mannitol fermentation and pigment forming ability of the mutant were more severely impaired than the wild type and complemented strains.5. The S. aureus L-form bacteria had complete or partial loss of cell wall with pleomorphic shape and became Gram-negative. The typical S. aureus L-form colonies had "fried egg" morphology. The optimal conditions for L-form formation were20%sucrose,3.5%sodium chloride, and750-1000units of penicillin and33℃ Stationary phase cultures of S. aureus formed L-forms better than log phase cultures. Mutant library screens identified15mutants deficient in L-form formation and sequencing analysis identified mutations in8genes and in3intergenic regions. Real-time PCR analysis indicated that except gntK,7genes including glpF, glpK, NWMN0623, NWMN0843, NWMN0333, NWMN0872, and NWMN1269were preferentially expressed in L-forms compared to normal cell walled form （P<0.05）. The glpF mutant could restore to form L-form after complementation with glpF or even cultured in LIM containing1%glycerol. The persistence of glpF mutant to ampicillin and norfloxacin was lower than the wild type and its defect could be complemented by wild type glpF.Conclusions:1.NWMN₀037plays important roles in S. aureus persister formation, tolerance to stress, growth at low temperature, biofilm formation, and virulence. NWMN0037can down-regulate bacterial metabolism, secrete exotoxins and extracellular proteases, influence bacterial biosynthesis, evade immunity, regulation, metabolism, cell division, biofilm formation, ABC transport, and SOS response, which contribute to its functions mentioned before. NWMN₀037may be an ideal candidate for development of new drugs targeting persisters for improved treatment and for vaccine development to fight S. aureus infection.2. The pathways of energy production, iron homeostasis, transporters, DNA repair, membrane biogenesis, and biosynthesis are found to be involved in S. aureus L-form formation. Glycerol uptake is important for L-form formation and persistence in S. aureus.3. The LIM containing20%sucrose,3.5%sodium chloride,750-1000units of penicillin and33℃cultured temperature are optimal conditions for S. aureus L-form formation. Stationary phase cultures of S. aureus form L-forms better than log phase cultures.