ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis
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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.
KeywordsLow 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.
Written informed consent was obtained from all participants.
- 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
- 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
- 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
- 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
- 20.Hutton CA, Perugini MA, Gerrard JA (2007) Inhibition of lysine biosynthesis: an evolving antibiotic strategy. Mol Bio Syst 3(7):458–465Google Scholar
- 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
- 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
- 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
- 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
- 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