C.W.I. Douglas

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method.

Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the significance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the fimbrial gene (fimA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Coamplification of target and competitive templates yielded products of expected size and non-specific recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The findings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

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.

Role of sialidase in glycoprotein utilization by Tannerella forsythia

The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host–pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.

Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion.

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10-30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

The effects of stress on periodontal treatment: a longitudinal investigation using clinical and biological markers

AIM To investigate the effects of psychosocial stress on the outcome of non-surgical periodontal treatment (NPT). METHODS Patients were categorized as stressed or unstressed, and the degree of stress was measured. One deep bleeding and one deep non-bleeding site ≥6 mm were selected in each patient for detailed investigation, and the clinical parameters were recorded before and at 6 months after NPT. Elastase and C-terminal teleopeptide of type I collagen (ICTP) were measured in gingival crevicular fluid (GCF) samples at both intervals. RESULTS The baseline, clinical parameters and biological markers were similar in both stressed and unstressed groups, other than for GCF elastase levels, which were significantly higher in the stressed group of patients (p < 0.05). The effect of stress on the changes for clinical measurements and elastase levels in GCF was statistically significant for deep bleeding sites, with the response to treatment being poorer in the stressed group. The effects of smoking and the degree of stress were not statistically significant for any of the clinical or biological parameters (p > 0.05). CONCLUSIONS Patients under psychosocial stress had a poorer outcome following NPT. The assessment of psychosocial stress may be valuable in the holistic management of periodontal disease.

Acid production and growth by oral Lactobacillus species in vitro.

AIM To analyze the acid-producing and growth abilities of different oral Lactobacillus species. METHODS Thirty-nine oral clinical strains and type strains of Lactobacillus, representing nine species, including Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus mucosae, Lactobacillus oris, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus vaginalis were studied. Anaerobically-grown bacterial cells were inoculated overnight in de Man, Rogosa, and Sharpe broth containing 2% glucose (pH 7.0). Acid production and growth were measured at 0, 1.5, 3, 5, 7, and 24 h. RESULTS Lactobacillus salivarius, Lactobacillus rhamnosus, Lactobacillus casei/paracasei, and Lactobacillus plantarum grew rapidly and reached an optical density higher than other species. They also produced more acid than the others. Lactobacillus vaginalis showed the lowest rate of growth and acid production. These findings demonstrated that the different species of Lactobacillus showed different abilities to generate acid, allowing the species to be categorized into three groups: strongly, moderately, and weakly acidogenic. CONCLUSION There was variation in acid production and growth between the Lactobacillus species. The strongest acid producers were Lactobacillus salivarius, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei/paracasei, respectively. It seems possible that these species might play a more important role in caries development than the others.

Physiological adaptations of key oral bacteria.

Oral colonising bacteria are highly adapted to the various environmental niches harboured within the mouth, whether that means while contributing to one of the major oral diseases of caries, pulp infections, or gingival/periodontal disease or as part of a commensal lifestyle. Key to these infections is the ability to adhere to surfaces via a range of specialised adhesins targeted at both salivary and epithelial proteins, their glycans and to form biofilm. They must also resist the various physical stressors they are subjected to, including pH and oxidative stress. Possibly most strikingly, they have developed the ability to harvest both nutrient sources provided by the diet and those derived from the host, such as protein and surface glycans. We have attempted to review recent developments that have revealed much about the molecular mechanisms at work in shaping the physiology of oral bacteria and how we might use this information to design and implement new treatment strategies.

Binding and degradation of lactoferrin by and

The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via a high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species (P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis, is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had orginated from healthy or diseased oral sites.