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Dual stable isotopes enhance lipidomic studies in bacterial model organism Enterococcus faecalis

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Abstract

Dual stable isotope probes of deuterium oxide and 13C fatty acid were demonstrated to probe the lipid biosynthesis cycle of a Gram-positive bacterium Enterococcus faecalis. As external nutrients and carbon sources often interact with metabolic processes, the use of dual-labeled isotope pools allowed for the simultaneous investigation of both exogenous nutrient incorporation or modification and de novo biosynthesis. Deuterium was utilized to trace de novo fatty acid biosynthesis through solvent-mediated proton transfer during elongation of the carbon chain while 13C-fatty acids were utilized to trace exogenous nutrient metabolism and modification through lipid synthesis. Ultra-high-performance liquid chromatography high-resolution mass spectrometry identified 30 lipid species which incorporated deuterium and/or 13C fatty acid into the membrane. Additionally, MS2 fragments of isolated lipids identified acyl tail position confirming enzymatic activity of PlsY in the incorporation of the 13C fatty acid into membrane lipids.

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Data availability

All raw data are publicly available through the MetaboLights database MTBLS5449.

References

  1. Brewer W, Harrison J, Saito HE, Fozo EM. Induction of daptomycin tolerance in Enterococcus faecalis by Fatty Acid Combinations. Appl Environ Microbiol. 2020;86(20):e01178-20.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Harp JR, Saito HE, Bourdon AK, Reyes J, Arias CA, Campagna SR, et al. Exogenous fatty acids protect Enterococcus faecalis from daptomycin-induced membrane stress independently of the response regulator LiaR. Appl Environ Microbiol. 2016;82(14):4410–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Saito HE, Harp JR, Fozo EM. Enterococcus faecalis Responds to Individual Exogenous Fatty Acids Independently of Their Degree of Saturation or Chain Length. Appl Environ Microbiol. 2017;84(1):e01633-17.

  4. Iram SH, Cronan JE. The & #x3b2;-Oxidation Systems of Escherichia coli and Salmonella enterica Are Not Functionally Equivalent. J Bacteriol. 2006;188(2):599–608.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hines KM, Waalkes A, Penewit K, Holmes EA, Salipante SJ, Werth BJ, et al. Characterization of the mechanisms of daptomycin resistance among Gram-positive bacterial pathogens by multidimensional lipidomics. mSphere. 2017;2(6):e00492-17.

  6. Rashid R, Cazenave-Gassiot A, Gao IH, Nair ZJ, Kumar JK, Gao L, et al. Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis. PLoS ONE. 2017;12(4): e0175886.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Zhang Y-M, Rock CO. Membrane lipid homeostasis in bacteria. Nat Rev Microbiol. 2008;6(3):222–33.

    Article  PubMed  Google Scholar 

  8. Zhu L, Zou Q, Cao X, Cronan JE. Enterococcus faecalis encodes an atypical auxiliary acyl carrier protein required for efficient regulation of fatty acid synthesis by exogenous fatty acids. mBio. 2019;10(3):e00577-19.

  9. White SW, Zheng J, Zhang Y-M, Rock CO. The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem. 2005;74(1):791–831.

    Article  CAS  PubMed  Google Scholar 

  10. Woodall BM, Harp JR, Brewer WT, Tague ED, Campagna SR, Fozo EM. Enterococcus faecalis readily adapts membrane phospholipid composition to environmental and genetic perturbation. Front Microbiol. 2021;12.

  11. Fozo EM, Rucks EA. The making and taking of lipids: the role of bacterial lipid synthesis and the harnessing of host lipids in bacterial pathogenesis. Adv Microb Physiol. 2016;69:51–155.

    Article  CAS  PubMed  Google Scholar 

  12. Mishra NN, Bayer AS, Tran TT, Shamoo Y, Mileykovskaya E, Dowhan W, et al. Daptomycin resistance in Enterococci is associated with distinct alterations of cell membrane phospholipid content. PLoS ONE. 2012;7(8): e43958.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jang C, Chen L, Rabinowitz JD. Metabolomics and isotope tracing. Cell. 2018;173(4):822–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dong W, Rawat ES, Stephanopoulos G, Abu-Remaileh M. Isotope tracing in health and disease. Curr Opin Biotechnol. 2022;76:102739.

    Article  CAS  PubMed  Google Scholar 

  15. Triebl A, Wenk MR. Analytical considerations of stable isotope labelling in lipidomics. Biomolecules. 2018;8(4):151.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Postle AD, Hunt AN. Dynamic lipidomics with stable isotope labelling. J Chromatogr B. 2009;877(26):2716–21.

    Article  CAS  Google Scholar 

  17. Zhang T, Muraih JK, Tishbi N, Herskowitz J, Victor RL, Silverman J, et al. Cardiolipin prevents membrane translocation and permeabilization by daptomycin. J Biol Chem. 2014;289(17):11584–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang Z, Chen L, Liu L, Su X, Rabinowitz JD. Chemical basis for deuterium labeling of fat and NADPH. J Am Chem Soc. 2017;139(41):14368–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kim J, Kang D, Lee SK, Kim T-Y. Deuterium oxide labeling for global omics relative quantification: application to lipidomics. Anal Chem. 2019;91(14):8853–63.

    Article  CAS  PubMed  Google Scholar 

  20. Wegener G, Kellermann MY, Elvert M. Tracking activity and function of microorganisms by stable isotope probing of membrane lipids. Curr Opin Biotechnol. 2016;41:43–52.

    Article  CAS  PubMed  Google Scholar 

  21. Hochuli M, Szyperski T, Wüthrich K. Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium. J Biomol NMR. 2000;17(1):33–42.

    Article  CAS  PubMed  Google Scholar 

  22. Wilkinson DJ, Cegielski J, Phillips BE, Boereboom C, Lund JN, Atherton PJ, et al. Internal comparison between deuterium oxide (D2O) and L-[ring-13C6] phenylalanine for acute measurement of muscle protein synthesis in humans. Physiol Rep. 2015;3(7):e12433.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Malaisse WJ, Ladrière L, Verbruggen I, Willem R. Metabolism of d-[1-13C]fructose, d-[2-13C]fructose, and d-[6-13C]fructose in rat hepatocytes incubated in the presence of H2O or D2O. Mol Genet Metab. 2002;75(2):162–7.

    Article  CAS  PubMed  Google Scholar 

  24. Naser FJ, Jackstadt MM, Fowle-Grider R, Spalding JL, Cho K, Stancliffe E, et al. Isotope tracing in adult zebrafish reveals alanine cycling between melanoma and liver. Cell Metab. 2021;33(7):1493-504.e5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fu X, Deja S, Fletcher JA, Anderson NN, Mizerska M, Vale G, et al. Measurement of lipogenic flux by deuterium resolved mass spectrometry. Nat Commun. 2021;12(1):3756.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Abranches J, Martinez AR, Kajfasz JK, Chávez V, Garsin DA, Lemos JA. The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis. J Bacteriol. 2009;191(7):2248–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gant TG. Using deuterium in drug discovery: leaving the label in the drug. J Med Chem. 2014;57(9):3595–611.

    Article  CAS  PubMed  Google Scholar 

  28. Guan XL, Riezman I, Wenk MR, Riezman H. Yeast lipid analysis and quantification by mass spectrometry. Methods in enzymology. 470: Elsevier; 2010. p. 369–91.

  29. Murphy RC, Axelsen PH. Mass spectrometric analysis of long-chain lipids. Mass Spectrom Rev. 2011;30(4):579–99.

    Article  CAS  PubMed  Google Scholar 

  30. Zou Q, Dong H, Zhu L, Cronan JE. The Enterococcus faecalis FabT transcription factor regulates fatty acid biosynthesis in response to exogeneous fatty acids. Front Microbiol. 2022;13:877582.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ehrhardt CJ, Chu V, Brown T, Simmons TL, Swan BK, Bannan J, et al. Use of fatty acid methyl ester profiles for discrimination of Bacillus cereus T-strain spores grown on different media. Appl Environ Microbiol. 2010;76(6):1902–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sohlenkamp C, Geiger O. Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol Rev. 2016;40(1):133–59.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Dr. Hector Castro and the Biological and Small Molecule Mass Spectrometry Core at the University of Tennessee for the use of their facilities.

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Authors and Affiliations

  1. Department of Chemistry, University of Tennessee, Knoxville, TN, USA

    Brittni Woodall & Shawn R. Campagna

  2. Department of Microbiology, University of Tennessee, Knoxville, TN, USA

    Elizabeth M. Fozo

  3. Biological and Small Molecule Mass Spectrometry Core, University of Tennessee, Knoxville, TN, USA

    Shawn R. Campagna

Authors
  1. Brittni Woodall
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  2. Elizabeth M. Fozo
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  3. Shawn R. Campagna
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The manuscript was written through the contributions of all of the authors. All of the authors have given approval to the final version of the manuscript. These authors contributed equally.

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Correspondence to Shawn R. Campagna.

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Woodall, B., Fozo, E.M. & Campagna, S.R. Dual stable isotopes enhance lipidomic studies in bacterial model organism Enterococcus faecalis. Anal Bioanal Chem 415, 3593–3605 (2023). https://doi.org/10.1007/s00216-023-04750-3

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  • DOI: https://doi.org/10.1007/s00216-023-04750-3

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