1887

Abstract

Bacteria can survive high doses of antibiotics through stochastic phenotypic diversification. We present initial evidence that folate metabolism could be involved with the formation of persisters. The aberrant expression of the folate enzyme gene fau seems to reduce the incidence of persisters to antibiotics. Folate-impaired bacteria had a lower generation rate for persisters to the antibiotics ampicillin and ofloxacin. Persister bacteria were detectable from the outset of the exponential growth phase in the complex media. Gene expression analyses tentatively showed distinctive profiles in exponential growth at times when bacteria persisters were observed. Levels of persisters were assessed in bacteria with altered, genetically and pharmacologically, folate metabolism. This work shows that by disrupting folate biosynthesis and usage, bacterial tolerance to antibiotics seems to be diminished. Based on these findings there is a possibility that bacteriostatic antibiotics such as anti-folates could have a role to play in clinical settings where the incidence of antibiotic persisters seems to drive recalcitrant infections.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000722
2018-09-24
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/11/1432.html?itemId=/content/journal/micro/10.1099/mic.0.000722&mimeType=html&fmt=ahah

References

  1. Fauvart M, De Groote VN, Michiels J. Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J Med Microbiol 2011;60:699–709 [CrossRef][PubMed]
    [Google Scholar]
  2. Claudi B, Spröte P, Chirkova A, Personnic N, Zankl J et al. Phenotypic variation of Salmonella in host tissues delays eradication by antimicrobial chemotherapy. Cell 2014;158:722–733 [CrossRef][PubMed]
    [Google Scholar]
  3. Fisher RA, Gollan B, Helaine S. Persistent bacterial infections and persister cells. Nat Rev Microbiol 2017;15:453–464 [CrossRef][PubMed]
    [Google Scholar]
  4. Teuscher F, Gatton ML, Chen N, Peters J, Kyle DE et al. Artemisinin‐induced dormancy in plasmodium falciparum: duration, recovery rates, and implications in treatment failure. J Infect Dis 2010;202:1362–1368 [CrossRef][PubMed]
    [Google Scholar]
  5. WHO World Malaria Report 2016 World Health Organization; 2016
    [Google Scholar]
  6. Stover P, Schirch V. The metabolic role of leucovorin. Trends Biochem Sci 1993;18:102–106 [CrossRef][PubMed]
    [Google Scholar]
  7. Piironen V, Edelmann M, Kariluoto S, Bedo Z. Folate in wheat genotypes in the healthgrain diversity screen. J Agric Food Chem 2008;56:9726–9731 [CrossRef][PubMed]
    [Google Scholar]
  8. Hansen S, Lewis K, Vulić M. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother 2008;52:2718–2726 [CrossRef][PubMed]
    [Google Scholar]
  9. Ren D, Bedzyk LA, Thomas SM, Ye RW, Wood TK. Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol 2004;64:515–524 [CrossRef][PubMed]
    [Google Scholar]
  10. Field MS, Szebenyi DM, Perry CA, Stover PJ. Inhibition of 5,10-methenyltetrahydrofolate synthetase. Arch Biochem Biophys 2007;458:194–201 [CrossRef][PubMed]
    [Google Scholar]
  11. Goyer A, Collakova E, Díaz de La Garza R, Quinlivan EP, Williamson J et al. 5-Formyltetrahydrofolate is an inhibitory but well tolerated metabolite in Arabidopsis leaves. J Biol Chem 2005;280:26137–26142 [CrossRef][PubMed]
    [Google Scholar]
  12. Ogwang S, Nguyen HT, Sherman M, Bajaksouzian S, Jacobs MR et al. Bacterial conversion of folinic acid is required for antifolate resistance. J Biol Chem 2011;286:15377–15390 [CrossRef][PubMed]
    [Google Scholar]
  13. Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol 2013;188:51–62 [CrossRef][PubMed]
    [Google Scholar]
  14. Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 2014;510:298–302 [CrossRef][PubMed]
    [Google Scholar]
  15. Orman MA, Brynildsen MP. Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob Agents Chemother 2013;57:3230–3239 [CrossRef][PubMed]
    [Google Scholar]
  16. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006;2:2006.0008 [CrossRef][PubMed]
    [Google Scholar]
  17. Sambrook J, Green M. Molecular cloning. In A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 2012
    [Google Scholar]
  18. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 2004;230:13–18 [CrossRef][PubMed]
    [Google Scholar]
  19. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611–622 [CrossRef][PubMed]
    [Google Scholar]
  20. Grenier F, Matteau D, Baby V, Rodrigue S. Complete Genome Sequence of Escherichia coli BW25113. Genome Announc 2014;2:e01038-14 [CrossRef][PubMed]
    [Google Scholar]
  21. Zhou K, Zhou L, Lim Q', Zou R, Stephanopoulos G et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol 2011;12:18 [CrossRef][PubMed]
    [Google Scholar]
  22. R Core Team R: A Language and Environment for Statistical Computing Vienna, Austria: R Foundation for Statistical Computing; 2016
    [Google Scholar]
  23. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS One 2016;10:1–13
    [Google Scholar]
  24. S, Josse J, Husson F. Factominer: an R package for multivariate analysis. J Stat Softw 2008;25:1–18 [CrossRef]
    [Google Scholar]
  25. Verstraeten N, Knapen W, Fauvart M, Michiels J. A historical perspective on bacterial persistence. Methods Mol Biol 2016;1333:3–13 [CrossRef][PubMed]
    [Google Scholar]
  26. Moyed HS, Bertrand KP. hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 1983;155:768–775[PubMed]
    [Google Scholar]
  27. Moyed HS, Broderick SH. Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 1986;166:399–403 [CrossRef][PubMed]
    [Google Scholar]
  28. Sezonov G, Joseleau-Petit D, D'Ari R. Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 2007;189:8746–8749 [CrossRef][PubMed]
    [Google Scholar]
  29. Gangan MS, Athale CA. Threshold effect of growth rate on population variability of Escherichia coli cell lengths. R Soc Open Sci 2017;4:160417 [CrossRef][PubMed]
    [Google Scholar]
  30. Torrey HL, Keren I, Via LE, Lee JS, Lewis K. High persister mutants in Mycobacterium tuberculosis. PLoS One 2016;11:e0155127 [CrossRef][PubMed]
    [Google Scholar]
  31. Vilchèze C, Hartman T, Weinrick B, Jain P, Weisbrod TR et al. Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2017;114:4495–4500 [CrossRef][PubMed]
    [Google Scholar]
  32. Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR. The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 2002;184:3909–3916 [CrossRef][PubMed]
    [Google Scholar]
  33. Bigger J. Treatment of staphylococcal infections with penicillin by intermittent sterilisation. The Lancet 1944;244:497–500 [CrossRef]
    [Google Scholar]
  34. Lewis K, Cells P. Persister cells. Annu Rev Microbiol 2010;64:357–372 [CrossRef]
    [Google Scholar]
  35. Shah D, Zhang Z, Khodursky A, Kaldalu N, Kurg K et al. Persisters: a distinct physiological state of E. coli. BMC Microbiol 2006;6:53 [CrossRef][PubMed]
    [Google Scholar]
  36. Luidalepp H, Jõers A, Kaldalu N, Tenson T. Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J Bacteriol 2011;193:3598–3605 [CrossRef][PubMed]
    [Google Scholar]
  37. Amato SM, Brynildsen MP. Persister heterogeneity arising from a single metabolic stress. Curr Biol 2015;25:2090–2098 [CrossRef][PubMed]
    [Google Scholar]
  38. Harms A, Maisonneuve E, Gerdes K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science 2016;354:aff4268 [CrossRef][PubMed]
    [Google Scholar]
  39. Jõers A, Kaldalu N, Tenson T. The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. J Bacteriol 2010;192:3379–3384 [CrossRef][PubMed]
    [Google Scholar]
  40. Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science 2004;305:1622–1625 [CrossRef][PubMed]
    [Google Scholar]
  41. Elowitz MB, Levine AJ, Siggia ED, Swain PS. Stochastic gene expression in a single cell. Science 2002;297:1183–1186 [CrossRef][PubMed]
    [Google Scholar]
  42. Skarstad K, Steen HB, Boye E. Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol 1983;154:656–662[PubMed]
    [Google Scholar]
  43. Tibbetts AS, Appling DR. Compartmentalization of mammalian folate-mediated one-carbon metabolism. Annu Rev Nutr 2010;30:57–81 [CrossRef][PubMed]
    [Google Scholar]
  44. Khodursky A, Guzmán EC, Hanawalt PC. Thymineless death lives on: new insights into a classic phenomenon. Annu Rev Microbiol 2015;69:247–263 [CrossRef][PubMed]
    [Google Scholar]
  45. Cameron DR, Shan Y, Zalis EA, Isabella V, Lewis K. A genetic determinant of persister cell formation in bacterial pathogens. J Bacteriol 2018;200:e00303-18 [CrossRef][PubMed]
    [Google Scholar]
  46. Pu Y, Zhao Z, Li Y, Zou J, Ma Q et al. Enhanced efflux activity facilitates drug tolerance in dormant bacterial cells. Mol Cell 2016;62:284–294 [CrossRef][PubMed]
    [Google Scholar]
  47. Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 2011;145:39–53 [CrossRef][PubMed]
    [Google Scholar]
  48. Kim JS, Heo P, Yang TJ, Lee KS, Cho DH et al. Selective killing of bacterial persisters by a single chemical compound without affecting normal antibiotic-sensitive cells. Antimicrob Agents Chemother 2011;55:5380–5383 [CrossRef][PubMed]
    [Google Scholar]
  49. Pan J, Bahar AA, Syed H, Ren D. Reverting antibiotic tolerance of Pseudomonas aeruginosa PAO1 persister cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one. PLoS One 2012;7:e45778 [CrossRef][PubMed]
    [Google Scholar]
  50. Pan J, Song F, Ren D. Controlling persister cells of Pseudomonas aeruginosa PDO300 by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one. Bioorg Med Chem Lett 2013;23:4648–4651 [CrossRef][PubMed]
    [Google Scholar]
  51. Que YA, Hazan R, Strobel B, Maura D, He J et al. A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria. PLoS One 2013;8:e80140 [CrossRef][PubMed]
    [Google Scholar]
  52. Starkey M, Lepine F, Maura D, Bandyopadhaya A, Lesic B et al. Identification of anti-virulence compounds that disrupt quorum-sensing regulated acute and persistent pathogenicity. PLoS Pathog 2014;10:e1004321 [CrossRef][PubMed]
    [Google Scholar]
  53. Allison KR, Brynildsen MP, Collins JJ. Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 2011;473:216–220 [CrossRef][PubMed]
    [Google Scholar]
  54. Gonen N, Assaraf YG. Antifolates in cancer therapy: structure, activity and mechanisms of drug resistance. Drug Resist Updat 2012;15:183–210 [CrossRef][PubMed]
    [Google Scholar]
  55. Estrada A, Wright DL, Anderson AC. Antibacterial antifolates: from development through resistance to the next generation. Cold Spring Harb Perspect Med 2016;6:a028324 [CrossRef][PubMed]
    [Google Scholar]
  56. Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH. Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 2007;23:213–222 [CrossRef][PubMed]
    [Google Scholar]
  57. Nzila A. The past, present and future of antifolates in the treatment of Plasmodium falciparum infection. J Antimicrob Chemother 2006;57:1043–1054 [CrossRef][PubMed]
    [Google Scholar]
  58. Hobbs CV, Anderson C, Neal J, Sahu T, Conteh S et al. Trimethoprim-Sulfamethoxazole prophylaxis during live malaria sporozoite immunization induces long-lived, homologous, and heterologous protective immunity against sporozoite challenge. J Infect Dis 2017;215:122–130 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000722
Loading
/content/journal/micro/10.1099/mic.0.000722
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Supplementary File 2

Supplementary File 3

Supplementary File 4

Supplementary File 5

PDF

Supplementary File 6

PDF

Supplementary File 7

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error