Metatranscriptomic analysis shows functional alterations in subgingival biofilm in young smokers with periodontitis: a pilot study

Authors

  • Renato Corrêa Viana Casarin Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba https://orcid.org/0000-0003-1743-5855
  • Rafaela Videira Clima da Silva Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba
  • Hélvis Enri de Sousa Paz Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba https://orcid.org/0000-0002-0619-2547
  • Camila Schmidt Stolf Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba https://orcid.org/0000-0002-5125-2326
  • Lucas Miguel Carvalho Universidade Estadual de Campinas, Centro de Pesquisas em Engenharias e Ciências Computacionais, Campinas https://orcid.org/0000-0002-8766-0452
  • Melline Fontes Noronha University of Illinois at Chicago, Research Resource Center, Research Informatics Core, Illinois https://orcid.org/0000-0003-0914-4679
  • Antonio Wilson Sallum Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba
  • Mabelle de Freitas Monteiro Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Prótese e Periodontia, Piracicaba https://orcid.org/0000-0001-9333-4349

DOI:

https://doi.org/10.1590/1678-7757-2024-003

Keywords:

Smoking, Periodontitis, RNA-Seq, Host-pathogen interactions, Gene expression, Oral microbiology, Non-invasive diagnostics

Abstract

This study aimed to assess the influence of smoking on the subgingival metatranscriptomic profile of young patients affected by stage III/IV and generalized periodontal disease. Methodology: In total, six young patients, both smokers and non-smokers (n=3/group), who were affected by periodontitis were chosen. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for case-control reporting were followed. Periodontal clinical measurements and subgingival biofilm samples were collected. RNA was extracted from the biofilm and sequenced via Illumina HiSeq. Differential expression analysis used Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and differentially expressed genes were identified using the Sleuth package in R, with a statistical cutoff of ≤0.05. Results: This study found 3351 KEGGs in the subgingival biofilm of both groups. Smoking habits altered the functional behavior of subgingival biofilm, resulting in 304 differentially expressed KEGGs between groups. Moreover, seven pathways were modulated: glycan degradation, galactose metabolism, glycosaminoglycan degradation, oxidative phosphorylation, peptidoglycan biosynthesis, butanoate metabolism, and glycosphingolipid biosynthesis. Smoking also altered antibiotic resistance gene levels in subgingival biofilm by significantly overexpressing genes related to beta-lactamase, permeability, antibiotic efflux pumps, and antibiotic-resistant synthetases. Conclusion: Due to the limitations of a small sample size, our data suggest that smoking may influence the functional behavior of subgingival biofilm, modifying pathways that negatively impact the behavior of subgingival biofilm, which may lead to a more virulent community.

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References

Nociti FH, Casati MZ, Duarte PM. Current perspective of the impact of smoking on the progression and treatment of periodontitis. Periodontol2000. 2015;67(1):187-210. doi: 10.1111/prd.12063

Champagne BM, Sebrié EM, Schargrodsky H, Pramparo P, Boissonnet C, Wilson E. Tobacco smoking in seven Latin American cities: the CARMELA study. Tob Control. 2010;19(6):457-62. doi: 10.1136/tc.2009.031666

Graetz C, Sälzer S, Plaumann A, Schlattmann P, Kahl M, Springer C, et al. Tooth loss in generalized aggressive periodontitis: prognostic factors after 17 years of supportive periodontal treatment. J Clin Periodonto.l 2017;44(6):612–9. doi: 10.1111/jcpe.12725

Kanmaz B, Lappin DF, Nile CJ, Buduneli N. Effects of smoking on non‐surgical periodontal therapy in patients with periodontitis Stage III or IV, and Grade C. J Periodontol. 2019;91(4):442–53. doi: 10.1002/JPER.19-0141

Mason MR, Preshaw PM, Nagaraja HN, Dabdoub SM, Rahman A, Kumar PS. The subgingival microbiome of clinically healthy current and never smokers. ISME J. 2014;9(1):268-72. doi: 10.1038/ismej.2014.114

Shah SA, Ganesan SM, Varadharaj S, Dabdoub SM, Walters JD, Kumar PS. The making of a miscreant: tobacco smoke and the creation of pathogen-rich biofilms. NPJ Biofilms Microbiomes. 2017;3:26. doi: 10.1038/s41522-017-0033-2

Duran‐Pinedo AE. Metatranscriptomic analyses of the oral microbiome. Periodontol 2000. 2020;85(1):28-45. doi: 10.1111/prd.12350

Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol. 2018;89(1):S159-72. doi: 10.1002/JPER.18-0006

Tong J, Zhao W, Lv H, Li W, Chen Z, Zhang C. Transcriptomic profiling in human decidua of severe preeclampsia detected by RNA sequencing. J Cell Biochem. 2018;119(1):607-15. doi: 10.1002/jcb.26221

Gómez MA, Belew AT, Navas A, Rosales-Chilama M, Murillo J, Dillon LA, et al. Early leukocyte responses in ex-vivo models of healing and non-healing human Leishmania (Viannia) panamensis infections. Frontiers in Cellular and Infection Microbiology. 2021;11:687607. doi: 10.3389/fcimb.2021.687607

von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP; STROBE Initiative. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008;61(4):344-9. doi: 10.1016/j.jclinepi.2007.11.008

Miller PD Jr, McEntire ML, Marlow NM, Gellin RG. An evidenced-based scoring index to determine the periodontal prognosis on molars. J Periodontol. 2014;85(2):214-25. doi: 10.1902/jop.2013.120675

Ainamo J, Bay I. Problems and proposals for recording gingivitis and plaque. Int Dent J. 1975;25(4):229-35.

Mühlemann HR, Son S. Gingival sulcus bleeding: a leading symptom in initial gingivitis. Helv Odontol Acta. 1971;15(2):107-13.

Andrews S. FastQC: a quality control tool for high throughput sequence data [Software]. Cambridge: Babraham Bioinformatics; 2010. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-20. doi: 10.1093/bioinformatics/btu170

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-9. doi: 10.1038/nmeth.1923

Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics. 2012;28(24):3211-7. doi: 10.1093/bioinformatics/bts611

Escapa IF, Huang Y, Chen T, Lin M, Kokaras A, Dewhirst FE, et al. Construction of habitat-specific training sets to achieve species-level assignment in 16S rRNA gene datasets. Microbiome. 2020;8(1):65. doi: 10.1186/s40168-020-00841-w

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011 May 15;29(7):644-52. doi: 10.1038/nbt.1883

Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34(5):525–7. doi: 10.1038/nbt.3519

Pimentel H, Bray NL, Puente S, Melsted P, Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017;14(7):687-90. doi: 10.1038/nmeth.4324

Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2015;44(D1):D457-62. doi: 10.1093/nar/gkv1070

Kim J, Kim MS, Koh AY, Xie Y, Zhan X. FMAP: Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies. BMC Bioinformatics. 2016;17(1). doi: 10.1186/s12859-016-1278-0

Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017;45(D1):D566-73. doi: 10.1093/nar/gkw1004

Ching T, Huang S, Garmire LX. Power analysis and sample size estimation for RNA-Seq differential expression. RNA. 2014;20(11):1684-96. doi: 10.1261/rna.046011.114

Kulkarni R, Antala S, Wang A, Amaral FE, Rampersaud R, LaRussa SJ, et al. Cigarette smoke increases Staphylococcus aureus biofilm formation via oxidative stress. Infect Immun. 2012;80(11):3804-11.10.1128/IAI.00689-12

Jia YJ, Liao Y, He YQ, Zheng MQ, Tong XT, Xue WQ, et al. Association between oral microbiota and cigarette smoking in the chinese population. Front Cell Infect Microbiol. 2021;11:658203. doi: 10.3389/fcimb.2021.658203

Wu J, Peters BA, Dominianni C, Zhang Y, Pei Z, Yang L, et al. Cigarette smoking and the oral microbiome in a large study of American adults. The ISME Journal. 2016;10(10):2435–46. doi: 10.1038/ismej.2016.37

Schwarz MG, Antunes D, Corrêa PR, Silva-Gonçalves AJ, Malaga W, Caffarena ER, et al. Mycobacterium tuberculosis and M. bovis BCG

Moreau fumarate reductase operons produce different polypeptides that may be related to non-canonical functions. Front Microbiol. 2021;11:624121. doi: 10.3389/fmicb.2020.624121

Zhang L, Liu C, Jiang Q, Yin Y. Butyrate in energy metabolism: there is still more to learn. Trends Endocrinol Metab. 2021;32(3):159-69. doi: 10.1016/j.tem.2020.12.003

Perinbam K, Chacko JV, Kannan A, Digman MA, Siryaporn A. A shift in central metabolism accompanies virulence activation in Pseudomonas aeruginosa. mBio. 2020;11(2):e02730-18. doi: 10.1128/mBio.02730-18

Hreha TN, Foreman S, Duran-Pinedo AE, Morris AP, Manucha W, Jones JR, et al. The three NADH dehydrogenases of Pseudomonas aeruginosa: their roles in energy metabolism and links to virulence. PLoS One. 2021;16(2):e0244142–2. doi: 10.1371/journal.pone.0244142

Cogo K, Calvi BM, Mariano FS, Franco GC, Gonçalves RB, Groppo FC. The effects of nicotine and cotinine on Porphyromonas gingivalis colonisation of epithelial cells. Archives of Oral Biology. 2009;54(11):1061–7. doi: 10.1016/j.archoralbio.2009.08.001

Cogo K, Andrade A, Labate CA, Bergamaschi CC, Berto LA, Franco GC, et al. Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine. J Periodontal Res. 2012;47(6):766-75. doi: 10.1111/j.1600-0765.2012.01494.x

Settem RP, Honma K, Nakajima T, Phansopa C, Roy S, Stafford GP, et al. A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression. Mucosal Immunol. 2013;6(2):415-26. doi: 10.1038/mi.2012.85

Chiu CY, Chou HC, Chang LC, Fan WL, Dinh MCV, Kuo YL, Chung WH, Lai HC, Hsieh WP, Su SC. Integration of metagenomics-metabolomics reveals specific signatures and functions of airway microbiota in mite-sensitized childhood asthma. Allergy. 2020;75(11):2846-2857. doi: 10.1111/all.14438

Lynskey NN, Reglinski M, Calay D, Siggins MK, Mason JC, Botto M, et al. Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase. PLoS Pathog. 2017;13(8):e1006493. doi: 10.1371/journal.ppat.1006493

Huang R, Li M, Ye M, Yang K, Xu X, Gregory RL. Effects of nicotine on Streptococcus gordonii growth, biofilm formation, and cell aggregation. Appl Environ Microbiol. 2014 Dec;80(23):7212-8. doi: 10.1128/AEM.02395-14

Silva RV, Rangel TP, Corrêa MG, Monteiro MF, Casati MZ, Ruiz KG, et al. Smoking negatively impacts the clinical, microbiological, and immunological treatment response of young adults with Grade C periodontitis. J Periodontal Res. 2022 Dec;57(6):1116-1126. doi: 10.1111/jre.13049

Altabtbaei K, Maney P, Ganesan SM, Dabdoub SM, Nagaraja HN, Kumar PS. Anna Karenina and the subgingival microbiome associated with periodontitis. Microbiome. 2021;9(1):97. doi: 10.1186/s40168-021-01056-3

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Published

2024-08-16 — Updated on 2024-10-01

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How to Cite

Casarin, R. C. V., Silva, R. V. C. da, Paz, H. E. de S., Stolf, C. S., Carvalho, L. M., Noronha, M. F., Sallum, A. W., & Monteiro, M. de F. (2024). Metatranscriptomic analysis shows functional alterations in subgingival biofilm in young smokers with periodontitis: a pilot study. Journal of Applied Oral Science, 32, e20240031. https://doi.org/10.1590/1678-7757-2024-003 (Original work published 2024)