Identification of key determinants in Porphyromonas gingivalis host-cell invasion assays.

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host-cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host-cell identity (cell line vs. primary), host-cell lysis method, and host-cell passage number had no significant effect on bacterial invasion. However, incubation time, host-cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5ß1 integrin. Interestingly, this may represent a population of cells with stem cell-like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported.

Investigation of a Novel Predictive Biomarker Profile for the Outcome of Periodontal Treatment.

BACKGROUND: An ability to predict the response to conventional non-surgical treatment of a periodontal site would be advantageous. However, biomarkers or tests devised to achieve this have lacked sensitivity. The aim of this study is to assess the ability of a novel combination of biomarkers to predict treatment outcome of patients with chronic periodontitis. METHODS: Gingival crevicular fluid (GCF) and subgingival plaque were collected from 77 patients at three representative sites, one healthy (probing depth [PD] ≤3 mm) and two diseased (PD ≥6 mm), at baseline and at 3 and 6 months after treatment. Patients received standard non-surgical periodontal treatment at each time point as appropriate. The outcome measure was improvement in probing depth of ≥2 mm. Concentrations of active enzymes (matrix metalloproteinase [MMP]-8, elastase, and sialidase) in GCF and subgingival plaque levels of Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum were analyzed for prediction of the outcome measure. RESULTS: Using threshold values of MMP-8 (94 ng/µL), elastase (33 ng/µL), sialidase (23 ng/µL), and levels of P. gingivalis (0.23%) and T. forsythia (0.35%), receiver operating characteristic curves analysis demonstrated that these biomarkers at baseline could differentiate healthy from diseased sites (sensitivity and specificity ≥77%). Furthermore, logistic regression showed that this combination of these biomarkers at baseline provided accurate predictions of treatment outcome (≥92%). CONCLUSION: The "fingerprint" of GCF enzymes and bacteria described here offers a way to predict the outcome of non-surgical periodontal treatment on a site-specific basis.

A pilot study of active enzyme levels in gingival crevicular fluid of patients with chronic periodontal disease.

AIM: To determine whether combinations of enzymes in gingival crevicular fluid (GCF) can act as improved biomarkers compared with single enzymes for predicting the outcome of treatment and also for diagnosing the clinical status of sites. METHODS: Thirty subjects with chronic periodontitis were recruited to a 12-month longitudinal pilot study. GCF samples from three representative sites: healthy (≤3 mm), deep non-bleeding (NB) (≥6 mm) and deep bleeding (DB) (≥6 mm) sites and clinical data were collected at baseline, 3 months, 6 months and 12 months following periodontal treatment. Active enzyme levels (MMP-8, cathepsin G, elastase, trypsin-like activity and sialidase) in GCF samples were assessed. The enzyme profiles and clinical data of each site were analysed for correlation and logistic regression was performed to find the predictive value of the active enzyme levels regarding the outcome of treatment. RESULTS: Twenty-two individuals completed the study. All active enzyme levels were significantly higher in diseased sites than healthy sites. Logistic regression showed that the combination of MMP8, elastase and sialidase provided accurate predictions of treatment outcome (88% for NB and 86% for DB), which was significantly better than each enzyme alone (61%). CONCLUSION: This pilot has suggested that combined active enzyme profiling could provide significant prediction of outcome of treatment.

Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in Streptococcus gordonii.

Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme alpha-amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP(A)TDAAT(R)NND. A novel strategy, based on the single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity.