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Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase.

ABSTRACT: Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the ?-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10-100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F₁F₀ ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6052003 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase.

Wang Ying Y Bojer Martin Saxtorph MS George Shilpa Elizabeth SE Wang Zhihao Z Jensen Peter Ruhdal PR Wolz Christiane C Ingmer Hanne H

Scientific reports 20180718 1

Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formatio ...[more]

PMID: 30022089

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Project description:BackgroundPersister cells comprise a phenotypic variant that shows extreme antibiotic tolerance resulting in treatment failures of bacterial infections. While this phenomenon has posed a great threat in public health, mechanisms underlying their formation in Staphylococcus aureus remain largely unknown. Increasing evidences of the presence of persister cells in recalcitrant infections underscores the great urgency to unravel the mechanism by which these cells develop. Previously, we characterized msaABCR operon that plays roles in regulation of virulence, biofilm development and antibiotic resistance. We also characterized the function of MsaB protein and showed that MsaB is a putative transcription factor that binds target DNA in response to nutrients availability.ResultsIn this study, we compared the number of persister cell in wild type, msaABCR deletion mutant and the complemented strain in two backgrounds USA300 LAC and Mu50. Herein, we report that msaABCR deletion mutant forms significantly less number of persister cells relative to wild type after challenge with various antibiotics in planktonic and biofilm growth conditions. Complementation of the msaABCR operon restored wild type phenotype. Combined antibiotic therapy along with msaABCR deletion significantly improves the killing kinetics of stationary phase and biofilm S. aureus cells. Transcriptomics analysis showed that msaABCR regulates several metabolic genes, transcription factors, transporters and enzymes that may play role in persister cells formation, which we seek to define in the future.ConclusionsThis study presented a new regulator, msaABCR operon, that is involved in the persister cells formation, which is a poorly understood in S. aureus. Indeed, we showed that msaABCR deletion significantly reduces the persister cells formation in all growth phases tested. Although, we have not yet defined the mechanism, we have shown that msaABCR regulates several metabolic, transporters, and extracellular proteases genes that have been previously linked with persister cells formation in other bacterial systems. Taken together, this study showed that inactivation of the msaABCR operon enhances the effectiveness of antibiotics for the treatment of S. aureus infections, especially in context of persister cells.

Project description:PhoU homologs are one of the determinant factors in the regulation of persister formation and phosphate metabolism in many bacterial species; however, the functions of PhoU homologs exhibit species-specific characteristics. The pathogenesis of Staphylococcus aureus is closely correlated with persister formation and virulence factors. The functions of two PhoU homologs, PhoU1 and PhoU2, in S. aureus are unclear yet. In this study, single- and double-deletion mutants of phoU1 and phoU2 were generated in strain USA500 2395. The ΔphoU1 or ΔphoU2 mutants displayed a change in persister formation and virulence compared to the parent strain; the persisters to vancomycin and levofloxacin were decreased at least 1,000-fold, and the number of intracellular bacteria surviving in the A549 cells for 24 h decreased to 82 or 85%. The α-hemolysin expression and activity were increased in the ΔphoU2 mutants. Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the ΔphoU1 or ΔphoU2 mutant vs. the wild type. Genes involved in carbon and pyruvate metabolism were up-regulated, and virulence genes and virulence regulatory genes were down-regulated, including type VII secretion system, serine protease, leukocidin, global regulator (sarA, rot), and the two-component signal transduction system (saeS). Correspondingly, the deletion of the phoU1 or phoU2 resulted in increased levels of intracellular pyruvate and ATP. Deletion of the phoU2, but not the phoU1, resulted in the up-regulation of inorganic phosphate transport genes and increased levels of intracellular inorganic polyphosphate. In conclusion, both PhoU1 and PhoU2 in S. aureus regulate virulence by the down-regulation of multiple virulence factors (type VII secretion system, serine protease, and leucocidin) and the persister generation by hyperactive carbon metabolism accompanied by increasing intracellular ATP. The results in S. aureus are different from what we have previously found in Staphylococcus epidermis, where only PhoU2 regulates biofilm and persister formation. The different functions of PhoU homologs between the two species of Staphylococcus warrant further investigation.

Project description:BackgroundStaphylococcus aureus is the most dominant human pathogen, responsible for a variety of chronic and severe infections. There is mounting evidence that persisters are associated with treatment failure and relapse of persistent infections. While some essential oils were reported to have antimicrobial activity against growing S. aureus, activity of essential oils against the stationary phase S. aureus enriched in persisters has not been investigated.MethodsIn this study, we evaluated the activity of 143 essential oils against both growing and stationary phase S. aureus by minimum inhibitory concentration (MIC) testing and by colony forming unit assay.ResultsWe identified 39 essential oils (Oregano, Cinnamon bark, Thyme white, Bandit "Thieves", Lemongrass (Cymbopogon flexuosus), Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa, Cinnamon leaf, Clove bud, Citronella, Geranium bourbon, Marjoram, Peppermint, Lemongrass, Cornmint, Elemi, Ho wood, Head ease, Lemon eucalyptus, Litsea cubeba, Myrrh, Parsley seed, Coriander oil, Dillweed, Hyssop, Neroli, Rosewood oil, Tea tree, Cajeput, Clove bud, Lavender, Sleep tight, Vetiver, Palo santo, Sage oil, Yarrow) at 0.5% (v/v) concentration, 10 essential oils (Cinnamon bark, Oregano, Thyme white, Bandit "Thieves", Lemongrass, Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa at 0.25% (v/v) concentration, and 7 essential oils (Oregano, Cinnamon bark, Thyme white, Lemongrass, Allspice, Amyris, Palmarosa at 0.125% (v/v) concentration to have high activity against stationary phase S. aureus with no visible growth on agar plates after five-day exposure. Among the 10 essential oils which showed high activity at 0.25% (v/v) concentration, 9 (Oregano, Cinnamon bark, Thyme white, Bandit "Thieves", Lemongrass, Health shield, Allspice, Palmarosa, Amyris showed higher activity than the known persister drug tosufloxacin, while Sandalwood oil had activity at a higher concentration. In Oregano essential oil combination studies with antibiotics, Oregano plus tosufloxacin (or levofloxacin, ciprofloxacin) and rifampin completely eradicated stationary phase S. aureus cells, but had no apparent enhancement for linezolid, vancomycin, sulfamethoxazole, trimethoprim, azithromycin or gentamicin.ConclusionsOur findings indicate that some essential oils have excellent activity against both growing and stationary phase S. aureus. Further studies are needed to identify the active components, evaluate safety, pharmacokinetics, and their activity to eradicate S. aureus infections in vivo.

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Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase.

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Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase.

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