Microbiome and Proteome Mapping in Periodontitis

Study Population: According to the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions, 3 systemically healthy and non-smoking patients with stage III, grade C periodontitis were included in this study. Clinical periodontal measurements included probing depth (PD), clinical attachment loss (CAL), the dichotomous scoring of bleeding on probing (BOP +/-), gingival index (GI), and plaque index (PI). All clinical parameters were recorded at six points (mesiobuccal, buccal, distobuccal, mesiopalatal palatal, and distopalatal) per tooth, except the 3rd molars, by a single investigator (B.A) using a manual periodontal probe. The percentage of radiographic bone loss (RBL) at the interproximal sites were calculated on the digital panoramic radiographs as the ratio of the distance between bone level and the cemento-enamel junction to the length of the root.

Sampling: Saliva, serum, gingival crevicular fluid (GCF) and subgingival plaque samples were collected 1 day following the clinical periodontal recordings. All samples were obtained in the morning between 8:30 am-10:30 am.

Firstly, the whole unstimulated saliva samples were collected. The participants were requested to refrain from eating, drinking (except water), chewing gum, brushing and flossing 2 hours before saliva collection. Then they were asked to let the saliva pool in their floor of the mouth and to allow the saliva to drain passively into a falcon tube, 5 minutes which was then stored directly at -80°C. On the day of analysis, 1 mL of thawed saliva was centrifuged at 4000 rpm at 4°C for 20 minutes.

To obtain serum samples, 5 ml of venous blood were taken from the antecubital vein by a standard venipuncture method. Serum was isolated by centrifugation at 1,500 g for 15 minutes at 4°C and then immediately frozen and stored at -80°C.

GCF was collected from the six sites (mesiobuccal, buccal, distobuccal, mesiopalatal, palatal, and distopalatal) per tooth, except the 3rd molars, via steril paper strips. A paper strip was placed inside the pocket till a slight resistance was encountered and was left there for 30 seconds. The absorbed fluid volume was measured with a calibrated electronic instrument. Six GCF strips from each tooth were placed directly into an empty Eppendorf tube and stored at -80°C until analysis. On the day of analysis, strips resuspended in 480 μL PBS in an Eppendorf tube. The sample was shaken overnight at 600 rpm at 4°C, then centrifuged for 60 minutes at 4000 rpm at 4°C.

Subgingival plaque was collected by inserting two standardized paper points (no:30) at six sites (mesiobuccal, buccal, distobuccal, mesiopalatal, palatal, and distopalatal) per tooth, except the 3rd molars. Two paper points were placed into the pocket and left place for 10 seconds. Paper points from each tooth per quadrant were placed directly into an empty Falcon tube and stored at -80°C until analysis. On the day of analysis, each plaque sample was obtained by pooling material from multiple teeth and resuspending it in 50 μL PBS per strip in a Falcon tube. The sample was shaken overnight at 600 rpm at 4°C, then vortexed for 1 minute on a benchtop vortexer, and sonicated for 1 min. It was then centrifuged for 60 min. at 4000 rpm at 4°C. 1 mL of the supernatant was collected for an additional centrifuge step at 16,100 x g for 10 minutes at 4°C before pellets were collected for further analysis.

DNA Extraction and Quantification: DNA was extracted from 1 mL of saliva, paper point extracts, and serum using the GenElute bacterial genomic DNA kit as per established protocols. Sample concentrations were quantified using a Qubit 4 fluorometer and the Qubit™ dsDNA HS Assay Kit.

Library Preparation and Sequencing: DNA libraries were prepared for next generation sequencing according to the established protocols. Sequencing was performed on an Illumina NovaSeq 6000 platform 2x150b.

Bioinformatics Analysis for Microbiota: This procedure included assessments of alpha diversity, beta diversity, and LDA effect size in the taxonomic analysis.

Proteomics analysis: Quantitative proteomics were done according to the established protocols. Peptides were separated using a Thermo Scientific EASY-nLC 1200 system with a 15 cm, 75 µm-diameter silica emitter packed with ReproSil-Pur C18-AQ resin. Protein identification was performed using Scaffold v4.0. MS data were searched with Mascot against a customized database, which included the Homo sapiens Uniprot database, a 260-sequence MS contaminants database, and reversed sequences as a decoy. Search results were imported into Scaffold for validation, with filters set at 3.0% protein false discovery rate with a minimum of 2 peptides, and a 1.0% peptide false discovery rate.

Functional Analysis of Regulated Proteins \& Statistical Analysis: We conducted enrichment analysis using the R software and the clusterProfiler package. Gene Ontology terms were identified from the 'Biological Process' database and ranked by statistical significance using hypergeometric distribution. Benjamini-Hochberg correction was applied, with significance thresholds set at p-value < 0.01 and q-value < 0.05.