European Spine Journal

, Volume 26, Issue 5, pp 1384–1400 | Cite as

ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis

  • S. Rajasekaran
  • Chitraa Tangavel
  • Siddharth N. Aiyer
  • Sharon Miracle Nayagam
  • M. Raveendran
  • Naveen Luke Demonte
  • Pramela Subbaiah
  • Rishi Kanna
  • Ajoy Prasad Shetty
  • K. Dharmalingam
Original Article


Study design

Proteomic and 16S rDNA analysis of disc tissues obtained in vivo.


To address the controversy of infection as an aetiology for disc disorders through protein profiling.

Summary of background data

There is raging controversy over the presence of bacteria in human lumbar discs in vivo, and if they represent contamination or infection. Proteomics can provide valuable insight by identifying proteins signifying bacterial presence and, also host defence response proteins (HDRPs), which will confirm infection.


22 discs (15-disc herniations (DH), 5-degenerate (DD), 2-normal in MRI (NM) were harvested intraoperatively and immediately snap frozen. Samples were pooled into three groups and proteins extracted were analysed with liquid chromatography-tandem mass spectrometry (LC–MS/MS). Post identification, data analysis was performed using Uniprotdb, Pantherdb, Proteome discoverer and STRING network. Authentication for bacterial presence was performed by PCR amplification of 16S rDNA.


LC–MS/MS analysis using Orbitrap showed 1103 proteins in DH group, compared to 394 in NM and 564 in DD. 73 bacterial specific proteins were identified (56 specific for Propionibacterium acnes; 17 for Staphylococcus epidermidis). In addition, 67 infection-specific HDRPs, unique or upregulated, such as Defensin, Lysozyme, Dermcidin, Cathepsin-G, Prolactin-Induced Protein, and Phospholipase-A2, were identified confirming presence of infection. Species-specific primers for P. acnes exhibited amplicons at 946 bp (16S rDNA) and 515 bp (Lipase) confirming presence of P. acnes in both NM discs, 11 of 15 DH discs, and all five DD discs. Bioinformatic search for protein–protein interactions (STRING) documented 169 proteins with close interactions (protein clustering co-efficient 0.7) between host response and degenerative proteins implying that infection may initiate degradation through Ubiquitin C.


Our study demonstrates bacterial specific proteins and host defence proteins to infection which strengthen the hypothesis of infection as a possible initiator of disc disease. These results can lead to a paradigm shift in our understanding and management of disc disorders.


Low back pain Disc degeneration Disc herniation Disc infections Proteomics LC–MS/MS Propionibacterium Staphylococcus rDNA Modic change Inflammation 



The authors would like to acknowledge Ms M Sujitha for assistance in LC–MS experiments, Dr. Velayudham Dinesh and Dr. Gopalkrishnan Chellappa for assistance in the bioinformatics. This study was supported by Ganga orthopaedic research and education foundation, Coimbatore, India.

Compliance with ethical standards

Conflict of interest

None of the authors have a conflict of interest.

Ethical statement

Written informed consent was obtained from all participants.


  1. 1.
    Stirling A, Worthington T, Rafiq M, Lambert PA, Elliott TS (2001) Association between sciatica and Propionibacterium acnes. Lancet 357(9273):2024–2025CrossRefPubMedGoogle Scholar
  2. 2.
    Albert HB, Sorensen JS, Christensen BS, Manniche C (2013) Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J 22(4):697–707CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Agarwal V, Golish SR, Alamin TF (2011) Bacteriologic culture of excised intervertebral disc from immunocompetent patients undergoing single level primary lumbar microdiscectomy. J Spinal Disord Tech 24(6):397–400CrossRefPubMedGoogle Scholar
  4. 4.
    Arndt J, Charles YP, Koebel C, Bogorin I, Steib JP (2012) Bacteriology of degenerated lumbar intervertebral disks. J Spinal Disord Tech 25(7):E211–E216CrossRefPubMedGoogle Scholar
  5. 5.
    Zhou Z, Chen Z, Zheng Y, Cao P, Liang Y, Zhang X, Wu W, Xiao J, Qiu S (2015) Relationship between annular tear and presence of Propionibacterium acnes in lumbar intervertebral disc. Eur Spine J 24(11):2496–2502CrossRefPubMedGoogle Scholar
  6. 6.
    Fritzell P, Bergström T, Welinder-Olsson C (2004) Detection of bacterial DNA in painful degenerated spinal discs in patients without signs of clinical infection. Eur Spine J 13(8):702–706CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kowalski TJ, Berbari EF, Huddleston PM, Steckelberg JM, Osmon DR (2007) Propionibacterium acnes vertebral osteomyelitis: seek and ye shall find? Clin Orthop Relat Res 1(461):25–30Google Scholar
  8. 8.
    Coscia MF, Denys GA, Wack MF (2016) Propionibacterium acnes, coagulase negative Staphylococcus, and the “biofilm-like” intervertebral disc. Spine (Phila Pa 1976) 41(24):1860–1865CrossRefGoogle Scholar
  9. 9.
    Capoor MN, Ruzicka F, Machackova T, Jancalek R, Smrcka M, Schmitz JE, Hermanova M, Sana J, Michu E, Baird JC, Ahmed FS (2016) Prevalence of Propionibacterium acnes in intervertebral discs of patients undergoing lumbar microdiscectomy: a prospective cross-sectional study. PLoS One 11(8):e0161676CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Urquhart DM, Zheng Y, Cheng AC, Rosenfeld JV, Chan P, Liew S, Hussain SM, Cicuttini FM (2015) Could low grade bacterial infection contribute to low back pain? A systematic review. BMC Med 13(1):1CrossRefGoogle Scholar
  11. 11.
    Chen Z, Cao P, Zhou Z, Yuan Y, Jiao Y, Zheng Y (2016) Overview: the role of Propionibacterium acnes in nonpyogenic intervertebral discs. Int Orthop 40:1291–1298CrossRefPubMedGoogle Scholar
  12. 12.
    Ganko R, Rao PJ, Phan K, Mobbs RJ (2015) Can bacterial infection by low virulent organisms be a plausible cause for symptomatic disc degeneration? A systematic review. Spine 40(10):E587–E592CrossRefPubMedGoogle Scholar
  13. 13.
    Wedderkopp N, Thomsen K, Manniche C, Kolmos HJ, Secher Jensen T, Leboeuf Yde C (2009) No evidence for presence of bacteria in modic type I changes. Acta Radiol 50(1):65–70CrossRefPubMedGoogle Scholar
  14. 14.
    Ben-Galim P, Rand N, Giladi M, Schwartz D, Ashkenazi E, Millgram M, Dekel S, Floman Y (2006) Association between sciatica and microbial infection: true infection or culture contamination? Spine 31(21):2507–2509CrossRefPubMedGoogle Scholar
  15. 15.
    Valkova N, Yunis R, Mak SK, Kang K, Kültz D (2005) Nek8 mutation causes overexpression of galectin-1, sorcin, and vimentin and accumulation of the major urinary protein in renal cysts of jck mice. Mol Cell Proteomics 4(7):1009–1018CrossRefPubMedGoogle Scholar
  16. 16.
    Ericsson C, Nistér M (2011) Protein extraction from solid tissue. In: Dillner J (ed) Methods in biobanking, vol 675. Humana Press, New york, pp 307–312Google Scholar
  17. 17.
    Anderson BL, Berry RW, Telser A (1983) A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000. Anal Biochem 132(2):365–375CrossRefPubMedGoogle Scholar
  18. 18.
    Fan H, Gulley ML (2001) DNA extraction from paraffin-embedded tissues. In: Killeen A (ed) Molecular pathology protocols, vol 49. Humana Press, New york, pp 1–4Google Scholar
  19. 19.
    Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hutton CA, Perugini MA, Gerrard JA (2007) Inhibition of lysine biosynthesis: an evolving antibiotic strategy. Mol Bio Syst 3(7):458–465Google Scholar
  21. 21.
    Hor L, Dobson RC, Downton MT, Wagner J, Hutton CA, Perugini MA (2013) Dimerization of bacterial diaminopimelate epimerase is essential for catalysis. J Biol Chem 288(13):9238–9248CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Bridges HR, Birrell JA, Hirst J (2011) The mitochondrial-encoded subunits of respiratory complex I (NADH: ubiquinone oxidoreductase): identifying residues important in mechanism and disease. Biochem Soc Trans 39(3):799–806CrossRefPubMedGoogle Scholar
  23. 23.
    Li Y, Park JS, Deng JH, Bai Y (2006) Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex. J Bioenerg Biomembr 38(5–6):283–291CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Li Y, Hodak M, Bernholc J (2015) Enzymatic mechanism of copper-containing nitrite reductase. Biochemistry 54(5):1233–1242CrossRefPubMedGoogle Scholar
  25. 25.
    Shiro Y (2012) Structure and function of bacterial nitric oxide reductases: nitric oxide reductase, anaerobic enzymes. Biochim Biophys Bioenerg 1817(10):1907–1913CrossRefGoogle Scholar
  26. 26.
    Fath MJ, Kolter R (1993) ABC transporters: bacterial exporters. Microbiol Rev 57(4):995–1017PubMedPubMedCentralGoogle Scholar
  27. 27.
    Saurin W, Hofnung M, Dassa E (1999) Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters. J Mol Evol 48(1):22–41CrossRefPubMedGoogle Scholar
  28. 28.
    Otaka E, Itoh T, Osawa S (1968) Ribosomal proteins of bacterial cells: strain-and species-specificity. J Mol Biol 33(1):93–107CrossRefPubMedGoogle Scholar
  29. 29.
    Begley TP, Kinsland C, Mehl RA, Osterman A, Dorrestein P (2001) The biosynthesis of nicotinamide adenine dinucleotides in bacteria. Vitam Horm 31(61):103–119CrossRefGoogle Scholar
  30. 30.
    Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12(24):3788–3796CrossRefPubMedGoogle Scholar
  31. 31.
    Taylor WE, Straus DB, Grossman AD, Burton ZF, Gross CA, Burgess RR (1984) Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase. Cell 38(2):371–381CrossRefPubMedGoogle Scholar
  32. 32.
    Edgerton M, Koshlukova SE, AraujoMW Patel RC, Dong J, Bruenn JA (2000) Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways. Antimicrob Agents Chemother 44(12):3310–3316CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, Longo DL, Taub DD, Oppenheim JJ (1996) Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 271(6):2935–2940CrossRefPubMedGoogle Scholar
  34. 34.
    Schittek B (2012) The multiple facets of dermcidin in cell survival and host defense. J Innate Immun 4(4):349–360CrossRefPubMedGoogle Scholar
  35. 35.
    Burian M, Schittek B (2015) The secrets of dermcidin action. Int J Med Microbiol 305(2):283–286CrossRefPubMedGoogle Scholar
  36. 36.
    Umadat V, Ihedioha O, Shiu R, Uzonna J, Myal Y (2013) The prolactin-inducible-protein (PIP): a regulatory molecule in adaptive and innate immunity. Open J Immunol 11:2013Google Scholar
  37. 37.
    Melrose J, Ghosh P, Taylor TK (1989) Lysozyme, a major low-molecular-weight cationic protein of the intervertebral disc, which increases with ageing and degeneration. Gerontology 35(4):173–180CrossRefPubMedGoogle Scholar
  38. 38.
    Anderson DG, Tannoury C (2005) Molecular pathogenic factors in symptomatic disc degeneration. Spine J 5(6):S260–S266CrossRefGoogle Scholar
  39. 39.
    Schroeder HW, Cavacini L (2010) Structure and function of immunoglobulins. J Allergy Clin Immunol 125(2):S41–S52CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Yu CY, Belt KT, Giles CM, Campbell RD, Porter RR (1986) Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity. EMBO J 5(11):2873PubMedPubMedCentralGoogle Scholar
  41. 41.
    Scuderi P, Nez PA, Duerr ML, Wong BJ, Valdez CM (1991) Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin. Cell Immunol 135(2):299–313CrossRefPubMedGoogle Scholar
  42. 42.
    Kepler CK, Ponnappan RK, Tannoury CA, Risbud MV, Anderson DG (2013) The molecular basis of intervertebral disc degeneration. Spine J 13(3):318–330CrossRefPubMedGoogle Scholar
  43. 43.
    Saal JS, Franson RC, Dobrow R, Saal JA, White AH, Goldthwaite N (1990) High levels of inflammatory phospholipase A2 activity in lumbar disc herniations. Spine 15(7):674–678CrossRefPubMedGoogle Scholar
  44. 44.
    Franson RC, Saal JS, Saal JA (1992) Human disc phospholipase A2 is inflammatory. Spine 17(6):S129–S132CrossRefPubMedGoogle Scholar
  45. 45.
    Nishihira J (2000) Macrophage migration inhibitory factor (MIF): its essential role in the immune system and cell growth. J Interferon Cytokine Res 20(9):751–762CrossRefPubMedGoogle Scholar
  46. 46.
    Calandra T, Roger T (2003) Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 3(10):791–800CrossRefPubMedGoogle Scholar
  47. 47.
    Schröder NW, Heine H, Alexander C, Manukyan M, Eckert J, Hamann L, Göbel UB, Schumann RR (2004) Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses. J Immunol 173(4):2683–2691CrossRefPubMedGoogle Scholar
  48. 48.
    Le Maitre CL, Freemont AJ, Hoyland JA (2004) Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol 204(1):47–54CrossRefPubMedGoogle Scholar
  49. 49.
    Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM (2000) Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 25(23):3005–3013CrossRefPubMedGoogle Scholar
  50. 50.
    Benbrook DM, Long A (2012) Integration of autophagy, proteasomal degradation, unfolded protein response and apoptosis. Exp Oncol 34(3):286–297PubMedGoogle Scholar
  51. 51.
    Roberts JL, Tavallai M, Nourbakhsh A, Fidanza A, Cruz-Luna T, Smith E, Siembida P, Plamondon P, Cycon KA, Doern CD, Booth L (2015) GRP78/Dna K is a target for nexavar/stivarga/votrient in the treatment of human malignancies, viral infections and bacterial diseases. J Cell Physiol 230(10):2552–2578CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Zhang YG, Guo X, Sun Z, Jia G, Xu P, Wang S (2010) Gene expression profiles of disc tissues and peripheral blood mononuclear cells from patients with degenerative discs. J Bone Miner Metab 28(2):209–219CrossRefPubMedGoogle Scholar
  53. 53.
    Minarowska A, Minarowski L, Karwowska A, Gacko M (2007) Regulatory role of cathepsin D in apoptosis. Folia Histochem Cytobiol 45(3):159–163PubMedGoogle Scholar
  54. 54.
    Streit M, Riccardi L, Velasco P, Brown LF, Hawighorst T, Bornstein P, Detmar M (1999) Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis. Proc Natl Acad Sci 96(26):14888–14893CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Oka C, Tsujimoto R, Kajikawa M, Koshiba-Takeuchi K, Ina J, Yano M, Tsuchiya A, Ueta Y, Soma A, Kanda H, Matsumoto M (2004) HtrA1 serine protease inhibits signaling mediated by Tgfβ family proteins. Development 131(5):1041–1053CrossRefPubMedGoogle Scholar
  56. 56.
    Heldin CH, Westermark B (1990) Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Regul 1(8):555PubMedPubMedCentralGoogle Scholar
  57. 57.
    Libert C, Brouckaert P, Fiers W (1994) Protection by alpha 1-acid glycoprotein against tumor necrosis factor-induced lethality. J Exp Med 180(4):1571–1575CrossRefPubMedGoogle Scholar
  58. 58.
    Moore DF, Rosenfeld MR, Gribbon PM, Winlove CP, Tsai CM (1997) Alpha-1-acid (AAG, orosomucoid) glycoprotein: interaction with bacterial lipopolysaccharide and protection from sepsis. Inflammation 21(1):69–82CrossRefPubMedGoogle Scholar
  59. 59.
    Roughley PJ, Mort JS (2014) The role of aggrecan in normal and osteoarthritic cartilage. J Exp Orthop 1(1):1CrossRefGoogle Scholar
  60. 60.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422(6928):198–207CrossRefPubMedGoogle Scholar
  61. 61.
    Aubin GG, Portillo ME, Trampuz A, Corvec S (2014) Propionibacterium acnes, an emerging pathogen: from acne to implant-infections, from phylotype to resistance. Medecine et maladies infectieuses 44(6):241–250CrossRefPubMedGoogle Scholar
  62. 62.
    Achermann Y, Goldstein EJ, Coenye T, Shirtliff ME (2014) Propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen. Clin Microbiol Rev 27(3):419–440CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Wei K, Tang DJ, He YQ, Feng JX, Jiang BL, Lu GT, Chen B, Tang JL (2007) hpaR, a putative marR family transcriptional regulator, is positively controlled by HrpG and HrpX and involved in the pathogenesis, hypersensitive response, and extracellular protease production of Xanthomonas campestris pathovar campestris. J Bacteriol 189(5):2055–2062CrossRefPubMedGoogle Scholar
  64. 64.
    Urban JP, Smith S, Fairbank JC (2004) Nutrition of the intervertebral disc. Spine 29(23):2700–2709CrossRefPubMedGoogle Scholar
  65. 65.
    Rajasekaran S, Bajaj N, Tubaki V, Kanna RM, Shetty AP (2013) ISSLS prize winner: the anatomy of failure in lumbar disc herniation: an in vivo, multimodal, prospective study of 181 subjects. Spine 38(17):1491–1500CrossRefPubMedGoogle Scholar
  66. 66.
    Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG (2002) Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 27(23):2631–2644CrossRefPubMedGoogle Scholar
  67. 67.
    Rajasekaran S, Babu JN, Arun R, Armstrong BR, Shetty AP, Murugan S (2004) ISSLS prize winner: a study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs. Spine 29(23):2654–2667CrossRefPubMedGoogle Scholar
  68. 68.
    Dessinioti C, Katsambas AD (2010) The role of Propionibacterium acnes in acne pathogenesis: facts and controversies. Clin Dermatol 28(1):2–7CrossRefPubMedGoogle Scholar
  69. 69.
    Burkhart CG, Burkhart CN, Lehmann PF (1999) Acne: a review of immunologic and microbiologic factors. Postgrad Med J 75(884):328–331CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Goldberg AL (2003) Protein degradation and protection against misfolded or damaged proteins. Nature 426(6968):895–899CrossRefPubMedGoogle Scholar
  71. 71.
    Li B, Dong Z, Wu Y, Zeng J, Zheng Q, Xiao B, Cai X, Xiao Z (2016) Association between lumbar disc degeneration and Propionibacterium acnes infection: clinical research and preliminary exploration of animal experiment. Spine 41(13):E764–E769CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • S. Rajasekaran
    • 1
  • Chitraa Tangavel
    • 2
  • Siddharth N. Aiyer
    • 1
  • Sharon Miracle Nayagam
    • 2
  • M. Raveendran
    • 3
  • Naveen Luke Demonte
    • 4
  • Pramela Subbaiah
    • 1
  • Rishi Kanna
    • 1
  • Ajoy Prasad Shetty
    • 1
  • K. Dharmalingam
    • 4
  1. 1.Department of Spine SurgeryGanga HospitalCoimbatoreIndia
  2. 2.Ganga Research CentreCoimbatoreIndia
  3. 3.Department of Plant BiotechnologyTamil Nadu Agricultural UniversityCoimbatoreIndia
  4. 4.Aravind Medical Research FoundationMaduraiIndia

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