Skip to main content
SpringerLink
Log in

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7–16.6% with 91.2–99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2–4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

EF:

Edema factor

ETx:

Edema toxin

LF:

Lethal factor

LTx:

Lethal toxin

PA:

Protective antigen

STD:

Standard

QC:

Quality control

stdev:

Standard deviation

mAb:

Monoclonal antibody

References

  1. Brossier F, Mock M. Toxins of Bacillus anthracis. Toxicon. 2001;39:1747–55.

    Article  CAS  PubMed  Google Scholar 

  2. Xu L, Frucht DM. Bacillus anthracis: a multi-faceted role for anthrax lethal toxin in thwarting host immune defenses. Int J Biochem Cell Biol. 2007;39(1):20–4. https://doi.org/10.1016/j.biocel.2006.08.010.

    Article  CAS  PubMed  Google Scholar 

  3. Leppla SH. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eukaryotic cells. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:189–98.

    CAS  PubMed  Google Scholar 

  4. Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A. 1982;79(10):3162–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Firoved AM, Miller GF, Moayeri M, Kakkar R, Shen Y, Wiggins JF, et al. Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol. 2005;167(5):1309–20. https://doi.org/10.1016/S0002-9440(10)61218-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Liu S, Moayeri M, Leppla SH. Anthrax lethal and edema toxins in anthrax pathogenesis. Trends Microbiol. 2014;22(6):317–25. https://doi.org/10.1016/j.tim.2014.02.012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. van der Goot G, Young JA. Receptors of anthrax toxin and cell entry. Mol Asp Med. 2009;30(6):406–12. https://doi.org/10.1016/j.mam.2009.08.007.

    Article  CAS  Google Scholar 

  8. Tournier JN, Rossi Paccani S, Quesnel-Hellmann A, Baldari CT. Anthrax toxins: a weapon to systematically dismantle the host immune defenses. Mol Asp Med. 2009;30(6):456–66. https://doi.org/10.1016/j.mam.2009.06.002.

    Article  CAS  Google Scholar 

  9. Turk BE. Manipulation of host signalling pathways by anthrax toxins. Biochem J. 2007;402(3):405–17. https://doi.org/10.1042/BJ20061891.

    Article  CAS  PubMed  Google Scholar 

  10. Boyer AE, Quinn CP, Woolfitt AR, Pirkle JL, McWilliams LG, Stamey KL, et al. Detection and quantification of anthrax lethal factor in serum by mass spectrometry. Anal Chem. 2007;79:8463–70.

    Article  CAS  PubMed  Google Scholar 

  11. Boyer AE, Gallegos-Candela M, Quinn CP, Woolfitt AR, Brumlow JO, Isbell K, et al. High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures. Anal Bioanal Chem. 2015;407(10):2847–58. https://doi.org/10.1007/s00216-015-8509-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Duriez E, Goossens PL, Becher F, Ezan E. Femtomolar detection of the anthrax edema factor in human and animal plasma. Anal Chem. 2009;81(14):5935–41. https://doi.org/10.1021/ac900827s.

    Article  CAS  PubMed  Google Scholar 

  13. Gottle M, Dove S, Kees F, Schlossmann J, Geduhn J, Konig B, et al. Cytidylyl and uridylyl cyclase activity of bacillus anthracis edema factor and Bordetella pertussis CyaA. Biochemistry. 2010;49(26):5494–503. https://doi.org/10.1021/bi100684g.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Duh SaC, J.D. Laboratory reference range values. Stedman’s online medical dictionary: University of Maryland; 2005.

  15. Gordon VM, Leppla SH, Hewlett EL. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect Immun. 1988;56(5):1066–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW. ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A. 1998;95:13899–904.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Leysath CE, Phillips DD, Crown D, Fattah RJ, Moayeri M, Leppla SH. Anthrax edema factor toxicity is strongly mediated by the N-end rule. PLoS One. 2013;8(8):e74474. https://doi.org/10.1371/journal.pone.0074474.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Boyer AE, Quinn CP, Hoffmaster AR, Kozel TR, Saile E, Marston CK, et al. Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques. Infect Immun. 2009;77(8):3432–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49–52.

    PubMed  PubMed Central  Google Scholar 

  20. Gallegos-Candela M, Boyer AE, Woolfitt AR, Brumlow J, Lins RC, Quinn CP, et al. Validated MALDI-TOF-MS method for anthrax lethal factor provides early diagnosis and evaluation of therapeutics. Anal Biochem. 2017;543:97–107. https://doi.org/10.1016/j.ab.2017.12.007.

    Article  CAS  PubMed  Google Scholar 

  21. HHS-FDA C, CVM. Guidance for industry: bioanalytical method validation. Rockville; 2018.

  22. Anaraki S, Addiman S, Nixon G, Krahe D, Ghosh R, Brooks T, et al. Investigations and control measures following a case of inhalation anthrax in East London in a drum maker and drummer, October 2008. Euro Surveill. 2008;13(51).

  23. Sprenkle MD, Griffith J, Marinelli W, Boyer AE, Quinn CP, Pesik NT, et al. Lethal factor and anti-protective antigen IgG levels associated with inhalation anthrax, Minnesota, USA. Emerg Infect Dis. 2014;20(2):310–4. https://doi.org/10.3201/eid2002.130245.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Walsh JJ, Pesik N, Quinn CP, Urdaneta V, Dykewicz CA, Boyer AE, et al. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. Clin Infect Dis. 2007;44(7):968–71.

    Article  PubMed  Google Scholar 

  25. Guo Q, Shen Y, Zhukovskaya NL, Florian J, Tang WJ. Structural and kinetic analyses of the interaction of anthrax adenylyl cyclase toxin with reaction products cAMP and pyrophosphate. J Biol Chem. 2004;279(28):29427–35. https://doi.org/10.1074/jbc.M402689200.

    Article  CAS  PubMed  Google Scholar 

  26. Lucey D. Bacillus anthracis (anthrax). In: G. Mandell JB, Dolin R, editors. Mandell, Douglas, and Bennet’s Principles and Practice of Infectious Diseases. Philadelphia: Churchill Livingstone; 2005. p. 2485–91.

    Google Scholar 

  27. Boyer AE, Gallegos-Candela M, Lins RC, Kuklenyik Z, Woolfitt A, Moura H, et al. Quantitative mass spectrometry for bacterial protein toxins--a sensitive, specific, high-throughput tool for detection and diagnosis. Molecules. 2011;16(3):2391–413.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ling WY, Marsh JM, LeMaire WJ. Adenosine-3′,5′-monophosphate in the plasma from human pregnancy. J Clin Endocrinol Metab. 1977;44(3):514–9. https://doi.org/10.1210/jcem-44-3-514.

    Article  CAS  PubMed  Google Scholar 

  29. Lin T. Plasma cyclic nucleotide levels in hyperthyroidism. Acta Endocrinol. 1979;90(1):62–8.

    Article  CAS  PubMed  Google Scholar 

  30. Hamet P, Stouder DA, Ginn HE, Hardman JG, Liddle GW. Studies of the elevated extracellular concentration of cyclic AMP in uremic man. J Clin Invest. 1975;56(2):339–45. https://doi.org/10.1172/JCI108098.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Marumo F, Sakai T, Shirataka M. A multivariate factor analysis of the high plasma concentration of cyclic AMP in patients with chronic renal failure. Int J Artif Organs. 1980;3(1):18–22.

    CAS  PubMed  Google Scholar 

  32. Schwarz W, Bock G, Hornstein OP. Plasma levels of cyclic nucleotides are elevated in atopic eczema. Arch Dermatol Res. 1987;279(Suppl):S59–62.

    Article  PubMed  Google Scholar 

  33. Hewlett EL, Gordon VM, McCaffery JD, Sutherland WM, Gray MC. Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule. J Biol Chem. 1989;264(32):19379–84.

    CAS  PubMed  Google Scholar 

  34. Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW. ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A. 1998;95(23):13899–904.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lehmann JS, Fouts DE, Haft DH, Cannella AP, Ricaldi JN, Brinkac L, et al. Pathogenomic inference of virulence-associated genes in Leptospira interrogans. PLoS Negl Trop Dis. 2013;7(10):e2468. https://doi.org/10.1371/journal.pntd.0002468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Matsunaga J, Lo M, Bulach DM, Zuerner RL, Adler B, Haake DA. Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host transition. Infect Immun. 2007;75(6):2864–74. https://doi.org/10.1128/IAI.01619-06.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rougeaux C, Becher F, Ezan E, Tournier JN, Goossens PL. In vivo dynamics of active edema and lethal factors during anthrax. Sci Rep. 2016;6:23346. https://doi.org/10.1038/srep23346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sirard JC, Mock M, Fouet A. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature. J Bacteriol. 1994;176(16):5188–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Molin FD, Fasanella A, Simonato M, Garofolo G, Montecucco C, Tonello F. Ratio of lethal and edema factors in rabbit systemic anthrax. Toxicon. 2008;52(7):824–8. https://doi.org/10.1016/j.toxicon.2008.08.011.

    Article  CAS  PubMed  Google Scholar 

  40. Pullan ST, Pearson TR, Latham J, Mason J, Atkinson B, Silman NJ, et al. Whole-genome sequencing investigation of animal-skin-drum-associated UK anthrax cases reveals evidence of mixed populations and relatedness to a US case. Microb Genom. 2015;1(5):e000039. https://doi.org/10.1099/mgen.0.000039.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported in part by Health and Human Services (HHS) Agencies: Biomedical Advanced Research and Development Authority (BARDA), Centers for Disease Control and Prevention (CDC) - Office of Public Health Preparedness and Response (OPHPR) and the National Institutes of Health (NIH) - Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

Author information

Author notes

  1. Clinton E. Leysath

    Present address: Center for Innovation in Advanced Development and Manufacturing, Texas A&M University Health Science Center, 8441 Riverside Parkway, Suite 3200, Bryan, TX, 77807, USA

Authors and Affiliations

  1. Battelle Atlanta Analytical Services, 2987 Clairmont Road NE, Suite 450, Atlanta, GA, 30329, USA

    Renato C. Lins & Judith O. Brumlow

  2. Centers for Disease Control and Prevention, 4770 Buford Highway Mailstop F-50, Atlanta, GA, 30341, USA

    Anne E. Boyer, Zsuzsanna Kuklenyik, Adrian R. Woolfitt, Maribel Gallegos-Candela & John R. Barr

  3. Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA, 30333, USA

    Jason Goldstein, Alex R. Hoffmaster, Conrad P. Quinn & Dennis A. Bagarozzi Jr

  4. Laboratory of Parasitic Diseases, NIAID, NIH, 9000 Rockville Pike, Bethesda, MD, 20892, USA

    Clinton E. Leysath & Stephen H. Leppla

  5. Laboratory of Infectious Diseases, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 9000 Rockville Pike, Bethesda, MD, 20892, USA

    Zhaochun Chen

Authors
  1. Renato C. Lins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anne E. Boyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zsuzsanna Kuklenyik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adrian R. Woolfitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jason Goldstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alex R. Hoffmaster
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maribel Gallegos-Candela
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Clinton E. Leysath
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhaochun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Judith O. Brumlow
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Conrad P. Quinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dennis A. Bagarozzi Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Stephen H. Leppla
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. John R. Barr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anne E. Boyer.

Ethics declarations

The study protocol was performed in 2006 and was approved by Battelle and the CDC Institutional Animal Care and Use Committees (IACUC) (CDC IACUC protocol no. 1459BOYMONX and Battelle MREF protocol no. 570).

Competing interests

The authors declare that they have no competing interests.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Use of trade names is for identification purposes only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Services, or the US Department of Health and Human Services.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 282 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lins, R.C., Boyer, A.E., Kuklenyik, Z. et al. Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax. Anal Bioanal Chem 411, 2493–2509 (2019). https://doi.org/10.1007/s00216-019-01730-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-01730-4

Keywords

Search

Navigation