Clinical Oral Investigations

, Volume 23, Issue 9, pp 3527–3534 | Cite as

Subgingival lipid A profile and endotoxin activity in periodontal health and disease

  • Alexander Strachan
  • Zoe Harrington
  • Clare McIlwaine
  • Matthew Jerreat
  • Louise A. Belfield
  • Aniko Kilar
  • Simon K. Jackson
  • Andrew Foey
  • Svetislav ZaricEmail author
Original Article



Regulation of lipopolysaccharide (LPS) chemical composition, particularly its lipid A domain, is an important, naturally occurring mechanism that drives bacteria-host immune system interactions into either a symbiotic or pathogenic relationship. Members of the subgingival oral microbiota can critically modulate host immuno-inflammatory responses by synthesizing different LPS isoforms. The objectives of this study were to analyze subgingival lipid A profiles and endotoxin activities in periodontal health and disease and to evaluate the use of the recombinant factor C assay as a new, lipid A-based biosensor for personalized, point-of-care periodontal therapy.

Materials and methods

Subgingival plaque samples were collected from healthy individuals and chronic periodontitis patients before and after periodontal therapy. Chemical composition of subgingival lipid A moieties was determined by ESI-Mass Spectrometry. Endotoxin activity of subgingival LPS extracts was assessed using the recombinant factor C assay, and their inflammatory potential was examined in THP-1-derived macrophages by measuring TNF-α and IL-8 production.


Characteristic lipid A molecular signatures, corresponding to over-acylated, bi-phosphorylated lipid A isoforms, were observed in diseased samples. Healthy and post-treatment samples were characterized by lower m/z peaks, related to under-acylated, hypo-phosphorylated lipid A structures. Endotoxin activity levels and inflammatory potentials of subgingival LPS extracts from periodontitis patients were significantly higher compared to healthy and post-treatment samples.


This is the first study to consider structure-function-clinical implications of different lipid A isoforms present in the subgingival niche and sheds new light on molecular pathogenic mechanisms of subgingival biofilm communities.

Clinical relevance

Subgingival endotoxin activity (determined by lipid A chemical composition) could be a reliable, bacterially derived biomarker and a risk assessment tool for personalized periodontal care.


Lipopolysaccharide Lipid A Subgingival microbiota Biomarker Periodontal diseases 



This study was supported by the Oral and Dental Research Trust’s GSK Research Grants Programme, the Dowager Countess Eleanor Peel Trust (219), and the New National Excellence Program of the Hungarian Ministry of Human Capacities (ÚNKP-17-4-III and NKFIH K-125275). Plymouth University Peninsula Schools of Medicine and Dentistry also acknowledges the support of the National Institute of Health Research Clinical Research Network (NIHR CRN).

Compliance with ethical standards

Conflict of interest

SZ has a patent on periodontitis biomarkers, publication number GB2549712. Author AS declares that he has no conflict of interest. Author ZH declares that she has no conflict of interest. Author CM declares that she has no conflict of interest. Author MJ declares that he has no conflict of interest. Author LB declares that she has no conflict of interest. Author AK declares that she has no conflict of interest. Author AF declares that he has no conflict of interest. Author SJ declares that he has no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Cafiero C, Matarasso S (2013) Predictive, preventive, personalised and participatory periodontology: ‘the 5Ps age’ has already started. EPMA J 4(1):16CrossRefGoogle Scholar
  2. 2.
    Silva N et al (2015) Host response mechanisms in periodontal diseases. J Appl Oral Sci 23(3):329–355CrossRefGoogle Scholar
  3. 3.
    Meuric V, le Gall-David S, Boyer E, Acuña-Amador L, Martin B, Fong SB, Barloy-Hubler F, Bonnaure-Mallet M (2017) Signature of microbial dysbiosis in periodontitis. Appl Environ Microbiol 83(14)Google Scholar
  4. 4.
    Taguchi H, Aono Y, Kawato T, Asano M, Shimizu N, Saigusa T (2015) Intragingival injection of Porphyromonas gingivalis-derived lipopolysaccharide induces a transient increase in gingival tumour necrosis factor-alpha, but not interleukin-6, in anaesthetised rats. Int J Oral Sci 7(3):155–160CrossRefGoogle Scholar
  5. 5.
    Jain S, Darveau RP (2010) Contribution of Porphyromonas gingivalis lipopolysaccharide to periodontitis. Periodontol 54(1):53–70CrossRefGoogle Scholar
  6. 6.
    Steimle A, Gronbach K, Beifuss B, Schäfer A, Harmening R, Bender A, Maerz JK, Lange A, Michaelis L, Maurer A, Menz S, McCoy K, Autenrieth IB, Kalbacher H, Frick JS (2016) Symbiotic gut commensal bacteria act as host cathepsin S activity regulators. J Autoimmun 75:82–95CrossRefGoogle Scholar
  7. 7.
    Fiusa MM et al (2015) Causes and consequences of coagulation activation in sepsis: an evolutionary medicine perspective. BMC Med 13:105CrossRefGoogle Scholar
  8. 8.
    Kramer CD, Genco CA (2017) Microbiota, immune subversion, and chronic inflammation. Front Immunol 8:255CrossRefGoogle Scholar
  9. 9.
    Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, Valentini S, Ferlenghi I, MacLennan CA, D'Oro U, Saul A, Gerke C (2014) Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants. J Biol Chem 289(36):24922–24935CrossRefGoogle Scholar
  10. 10.
    Coats SR, Pham TTT, Bainbridge BW, Reife RA, Darveau RP (2005) MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex. J Immunol 175(7):4490–4498CrossRefGoogle Scholar
  11. 11.
    Herath TD et al (2013) Tetra- and penta-acylated lipid A structures of Porphyromonas gingivalis LPS differentially activate TLR4-mediated NF-kappaB signal transduction cascade and immuno-inflammatory response in human gingival fibroblasts. PLoS One 8(3):e58496CrossRefGoogle Scholar
  12. 12.
    To TT et al (2015) Subgingival plaque in periodontal health antagonizes at Toll-like receptor 4 and inhibits E-selectin expression on endothelial cells. Infect Immun 84(1):120–126CrossRefGoogle Scholar
  13. 13.
    Coats SR, Jones JW, Do CT, Braham PH, Bainbridge BW, To TT, Goodlett DR, Ernst RK, Darveau RP (2009) Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1- and 4′-phosphatase activities. Cell Microbiol 11(11):1587–1599CrossRefGoogle Scholar
  14. 14.
    Foey AD, Crean S (2013) Macrophage subset sensitivity to endotoxin tolerisation by Porphyromonas gingivalis. PLoS One 8(7):e67955CrossRefGoogle Scholar
  15. 15.
    Hajishengallis G, Lamont RJ (2012) Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology. Mol Oral Microbiol 27(6):409–419CrossRefGoogle Scholar
  16. 16.
    Barros SP et al (2016) Gingival crevicular fluid as a source of biomarkers for periodontitis. Periodontology 2000 70(1):53–64CrossRefGoogle Scholar
  17. 17.
    Raetz CR et al (2007) Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 76:295–329CrossRefGoogle Scholar
  18. 18.
    Slocum C, Coats SR, Hua N, Kramer C, Papadopoulos G, Weinberg EO, Gudino CV, Hamilton JA, Darveau RP, Genco CA (2014) Distinct lipid a moieties contribute to pathogen-induced site-specific vascular inflammation. PLoS Pathog 10(7):e1004215CrossRefGoogle Scholar
  19. 19.
    Zenobia C, Hasturk H, Nguyen D, van Dyke TE, Kantarci A, Darveau RP (2014) Porphyromonas gingivalis lipid A phosphatase activity is critical for colonization and increasing the commensal load in the rabbit ligature model. Infect Immun 82(2):650–659CrossRefGoogle Scholar
  20. 20.
    Posch G, Andrukhov O, Vinogradov E, Lindner B, Messner P, Holst O, Schäffer C (2013) Structure and immunogenicity of the rough-type lipopolysaccharide from the periodontal pathogen Tannerella forsythia. Clin Vaccine Immunol 20(6):945–953CrossRefGoogle Scholar
  21. 21.
    Masoud H et al (1991) Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021-7. Eur J Biochem 200(3):775–781CrossRefGoogle Scholar
  22. 22.
    Asai Y, Makimura Y, Kawabata A, Ogawa T (2007) Soluble CD14 discriminates slight structural differences between lipid as that lead to distinct host cell activation. J Immunol 179(11):7674–7683CrossRefGoogle Scholar
  23. 23.
    John CM, Phillips NJ, Din R, Liu M, Rosenqvist E, Høiby EA, Stein DC, Jarvis GA (2016) Lipooligosaccharide structures of invasive and carrier isolates of Neisseria meningitidis are correlated with pathogenicity and carriage. J Biol Chem 291(7):3224–3238CrossRefGoogle Scholar
  24. 24.
    SenGupta S, Hittle LE, Ernst RK, Uriarte SM, Mitchell TC (2016) A Pseudomonas aeruginosa hepta-acylated lipid A variant associated with cystic fibrosis selectively activates human neutrophils. J Leukoc Biol 100(5):1047–1059CrossRefGoogle Scholar
  25. 25.
    Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS (2011) Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS Pathog 7(12):e1002454CrossRefGoogle Scholar
  26. 26.
    Socransky SS, Haffajee AD, Teles R, Wennstrom JL, Lindhe J, Bogren A, Hasturk H, van Dyke T, Wang X, Goodson JM (2013) Effect of periodontal therapy on the subgingival microbiota over a 2-year monitoring period. I. Overall effect and kinetics of change. J Clin Periodontol 40(8):771–780CrossRefGoogle Scholar
  27. 27.
    Feres M, Teles F, Teles R, Figueiredo LC, Faveri M (2016) The subgingival periodontal microbiota of the aging mouth. Periodontol 72(1):30–53CrossRefGoogle Scholar
  28. 28.
    Ding JL, Ho B (2010) Endotoxin detection--from limulus amebocyte lysate to recombinant factor C. Subcell Biochem 53:187–208CrossRefGoogle Scholar
  29. 29.
    Gutsmann T, Howe J, Zähringer U, Garidel P, Schromm AB, Koch MHJ, Fujimoto Y, Fukase K, Moriyon I, Martínez-de-Tejada G, Brandenburg K (2010) Structural prerequisites for endotoxic activity in the Limulus test as compared to cytokine production in mononuclear cells. Innate Immun 16(1):39–47CrossRefGoogle Scholar
  30. 30.
    Gronbach K, Flade I, Holst O, Lindner B, Ruscheweyh HJ, Wittmann A, Menz S, Schwiertz A, Adam P, Stecher B, Josenhans C, Suerbaum S, Gruber AD, Kulik A, Huson D, Autenrieth IB, Frick JS (2014) Endotoxicity of lipopolysaccharide as a determinant of T-cell-mediated colitis induction in mice. Gastroenterology 146(3):765–775CrossRefGoogle Scholar
  31. 31.
    Camelo-Castillo AJ et al (2015) Subgingival microbiota in health compared to periodontitis and the influence of smoking. Front Microbiol 6:119CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Medicine and DentistryUniversity of PlymouthPlymouthUK
  2. 2.Medical School, Institute of BioanalysisUniversity of PécsPécsHungary

Personalised recommendations