Homotypic biofilm structure of Porphyromonas gingivalis is affected by FimA type variations.

INTRODUCTION: Porphyromonas gingivalis is a periodontal pathogen whose long fimbriae (FimA) are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes. FimA variations were previously shown to be related to the onset and development of adult periodontitis in a general population, while FimA were recently found to be critical mediators of initial biofilm formation. However, it is unclear if FimA variations have effects on biofilm features. Here, we compare the characteristic structures of homotypic biofilms developed by P. gingivalis strains with different FimA types. METHODS: Biofilms were formed on saliva-coated glass bottom wells in phosphate-buffered saline and their structures were analysed using confocal laser scanning microscopy. Furthermore, the biovolumes of the biofilms were quantified with a three-dimensional fluorophotometric method. RESULTS: Biofilm structures formed by the six representative FimA-type strains apparently differed. Type I and Ib P. gingivalis formed biofilms with a dense basal monolayer and dispersed microcolonies, whereas those formed by types II, III and IV strains had markedly luxuriant biofilms filled with widely clumped and tall colonies, and their biovolumes were significantly greater than those of types I and Ib. These characteristic features were confirmed to be closely related to FimA type in assays that utilized fimA-substituted mutants from type I to II and those from type II to I. CONCLUSION: Our results suggest that FimA variations have effects on the structures of biofilms formed by P. gingivalis, which may be an important factor in the pathogenesis of periodontitis.

Relationship of Porphyromonas gingivalis with glycemic level in patients with type 2 diabetes following periodontal treatment.

INTRODUCTION: The aim of this study was to assess the relationship between serum glycemic levels and subgingival microbial profile alteration following periodontal treatment in patients with type 2 diabetes mellitus. METHODS: We studied 30 periodontitis patients with type 2 diabetes mellitus who received full-mouth subgingival debridement by analyzing their subgingival microbial profiles using a polymerase chain reaction method at baseline and various time-points for 12 months following treatment. Concurrently, probing pocket depth, bleeding on probing, and metabolic parameters, including glycated hemoglobin A1c (HbA1c), blood sugar level, C-reactive proteins, total cholesterol, triglyceride, and high-density and low-density lipoprotein cholesterol, were recorded. RESULTS: Periodontal conditions were significantly improved after treatment, and the occurrence rates of periodontal bacterial species, including Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Prevotella intermedia, were also reduced. Interestingly, P. gingivalis was detected more frequently in subjects with increased HbA1c values after periodontal treatment than in those patients with decreased HbA1c values. Furthermore, P. gingivalis with type II fimbriae was detected only in HbA1c-increased subjects, while improvements in HbA1c values were observed only in subjects without type II clones. CONCLUSIONS: These results suggest that glycemic level in diabetes is affected by the persistence of P. gingivalis, especially clones with type II fimbriae, in periodontal pockets.

Clinical assessment of oral malodor intensity expressed as absolute value using an electronic nose.

OBJECTIVES: In our previous study, scores determined via a multiple linear regression method (EN-MLR) involving an electronic nose provided objective halitosis-related measurements; however, this model afforded only relative expression exclusively. The objective of this investigation was to assess clinically oral malodor intensity expressed as an absolute value using an electronic nose. SUBJECTS AND METHODS: Sixty-six subjects were evaluated based on results of an actual organoleptic test (OLT), measurements of volatile sulfur compound (VSC) concentrations, a score representing malodor intensity (EN-MI) as the absolute value and EN-MLR measured with an electronic nose system. Oral health parameters were also examined. RESULTS: The OLT score served as a benchmark. The area under the receiver-operating characteristic (ROC) plots of EN-MI score (0.975) was significantly larger than that of log VSC (0.896) (P = 0.036); however, the area did not differ significantly from that of EN-MLR score (0.932). Percentage of teeth with pocket depth greater than or equal to 4 mm, tongue coating score and plaque control record displayed meaningful association with EN-MI score in multiple logistic regression analyses. CONCLUSION: Oral malodor intensity expressed as an absolute value employing an electronic nose may be a suitable method for clinical evaluation of oral malodor.

Effect of topical application of coenzyme Q10 on adult periodontitis.

Topical application of Coenzyme Q10 (CoQ10) to the periodontal pocket was evaluated with and without subgingival mechanical debridement. Ten male patients with adult periodontitis participated and 30 periodontal pockets were selected. During the first 3 weeks, the patients did not receive any periodontal therapy except the topical application of CoQ10. After the first 3-week period, root planning and subgingival scaling were performed in all sites. CoQ10 was applied in 20 of the pockets once a week for a period of 6 weeks. Soybean oil was applied to the remaining 10 sites as a control. In the first 3-week period, significant reductions in gingival crevicular fluid flow, probing depth and attachment loss were found only at experimental sites. After mechanical subgingival debridement, significant decreases in the plaque index, gingival crevicular fluid flow, probing depth and attachment loss were found both at experimental and control sites. However, significant improvements in the modified gingival index, bleeding on probing and peptidase activity derived from periodontopathic bacteria were observed only at experimental sites. These results suggest that topical application of CoQ10 improves adult periodontitis not only as a sole treatment but also in combination with traditional nonsurgical periodontal therapy.

The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis.

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21,500. The sequence of B. gingivalis SOD showed 44-51% homology with those for iron-specific SODs (Fe-SODs) and 40-45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65-74%) or Mn-SOD family (42-60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families.

Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis.

Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.

Inhibition of coaggregation between Porphyromonas gingivalis and Streptococcus oralis by fibrinogen fragments.

The localization of regions of fibrinogen that inhibit coaggregation between Porphyromonas gingivalis and Streptococcus oralis was investigated. The coaggregation was inhibited by A alpha and gamma chains, but not by B beta chain. The inhibitory activity of fragment D was more potent than that of fragment E. Some cyanogen bromide-treated fragments isolated from A alpha and gamma chains including the NH2-terminal 148-207 amino acid residues of A alpha chain (A alpha 148-207) and gamma 1-78 showed inhibitory activities. A alpha 148-207 was further digested with lysyl endopeptidase. A alpha 158-176 and A alpha 192-206 which contained four and two arginine residues, respectively, retained the inhibitory activities. When the arginine residues of these two peptides were modified by phenylglyoxal, the inhibitory activities were much reduced. These findings suggest that the arginine residues of some specific regions of fibrinogen may play an important role in the inhibition of the coaggregation.

Binding of a histidine-rich peptide to Porphyromonas gingivalis.

Porphyromonas gingivalis 381 cells were incubated with 125I-histidine-rich polypeptide (histatin) 5 in the presence or absence of unlabeled histatin 5, to evaluate the histatin-binding capacity of the cells. The binding of histatin 5 was rapid, reversible, saturable and specific. The number of histatin 5-binding sites per cell was 3,600, and the dissociation constant (Kd) was in the order of 10(-6) M. These findings suggest that histatin interacts with certain bacterial cells through specific binding sites on their surface, and will allow the development of a histatin radioreceptor assay.

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381.

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.

Interaction of Porphyromonas gingivalis with transferrin.

In this study, we characterized the binding of transferrin to Porphyromonas gingivalis using a classical receptor-binding assay, and examined the relationship between the binding and availability of transferrin for the growth of P. gingivalis. The binding of 125I-labeled human transferrin to P. gingivalis occurred rapidly, reversibly and specifically. Scatchard analysis yielded a Kd of 1.37 +/- 0.16 microM and an apparent number of 1.13 +/- 0.26 x 10(5) receptors per cell. The binding of transferrin was much increased when organisms were grown in iron-limited conditions. Among the species of black-pigmented anaerobic.rods, those strains of P. gingivalis which had high transferrin-binding activity exhibited unrestricted growth following the addition of transferrin to the hemin-free culture medium. On the other hand, the presence of transferrin in the culture medium did not support unrestricted growth of organisms that had low transferrin-binding activity. These results suggest that the binding of transferrin to P. gingivalis cells may be a preliminary step in iron acquisition, which allows them to survive in the healthy periodontal environment.

Effect of concentration of compounds containing iron on the growth of Porphyromonas gingivalis.

We examined the effect of the concentration of various types of iron molecules on the regulation of growth of Porphyromonas gingivalis. Bacterial growth was monitored spectrophotometrically. The hemin-depleted cells of P. gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron compounds. The relationship between the specific growth rate of organisms and the concentration of iron-containing compounds was determined. The value of Ks, a parameter analogous to the Michaelis-Menten constant, was estimated. P. gingivalis 381 showed a Ks value of 3.85, 4.91 and 0.0017 microM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support growth of P. gingivalis. These findings suggest that P. gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron-limited environment.

Binding of hemoglobin by Porphyromonas gingivalis.

In this study, we investigated whether Porphyromonas gingivalis can bind hemoglobin as an initial step in the acquisition of heme from hemoglobin. The binding of human hemoglobin by P. gingivalis cells was determined using [3H]hemoglobin. Hemoglobin binding occurred rapidly, reversibly and specifically. A Scatchard analysis of the binding data generated a linear plot, indicating a single population of binding proteins. The apparent Kd was 1.0 +/- 0.19 x 10(-6) M and there were 3.2 +/- 0.76 x 10(4) binding sites per cell. Hemoglobin binding was inhibited by unlabeled human hemoglobin but not by hemin and protoporphyrin IX. The binding was only partially inhibited by human serum albumin, transferrin, lactoferrin, catalase and cytochrome c. These results suggest that the ligand recognized by the binding protein may not be the heme moiety. The binding of hemoglobin considerably increased when the organisms were grown under hemin-limited conditions. Hemoglobin bound to outer membrane proteins extracted from P. gingivalis cells on a dot blot binding assay and binding ability was lost after heating bacterial proteins. These results suggest that P. gingivalis cells interact with human hemoglobin through specific binding sites on their surfaces as a preliminary step in iron acquisition.

Lactose synthase activity of galactosyltransferase from human parotid saliva.

Galactosyltransferase is human parotid saliva catalyzed the transfer of galactose from uridine 5'-diphospho (UDP)-galactose to D-glucose in the presence of exogenous alpha-lactalbumin. Some albumins other than alpha-lactalbumin did not have an effect on the appearance of lactosesynthesizing activity.

Changes in oxygen consumption in dog gingiva during induction of experimental periodontitis.

The purpose of this study was to examine changes in oxygen consumption in dog gingiva during induction of experimental periodontitis. The disease was induced in adult mongrel dogs during a 16-week period by placement of silk ligatures around selected teeth. The oxygen consumption rate of gingival tissue was determined in vivo by a non-invasive technique, tissue reflectance spectrophotometry. Changes in such clinical parameters as gingival index, plaque index, pocket depth, attachment level, and gingival crevicular fluid flow indicated acute inflammatory responses during the first three weeks after ligation, followed by the appearance of chronic inflammation during the remaining 13 weeks. The oxygen consumption rate increased during the first seven days after ligation and stayed near the maximum level for 2-7 weeks; this was followed by a gradual decrease during the final nine weeks. These results suggest that gingival oxygen consumption increases rapidly with the increase of acute inflammation responses and then decreases slightly with the gradual development of chronic inflammation. Positive correlations were observed between the oxygen consumption rate and other clinical indices. Thus, the tissue reflectance spectrophotometry is a new, useful method for objective, quantitative, and non-invasive assessment of gingival oxygen consumption.

Inhibitory effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis.

The effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis was studied by means of a turbidimetric assay. The co-aggregation activity was obtained from the maximum slope of the absorbance vs. time curve. Its dependence on pH, temperature, and ionic strength was examined, and the number of Bacteroides cells in relation to the number of Streptococcus cells resulting in optimal co-aggregation was established. Co-aggregation inhibition experiments showed that the co-aggregation activity was inhibited by l-arginine and l-lysine, although the activity was unaffected by the sugars tested. Human plasma and saliva were able to inhibit the co-aggregation in a dose-dependent reaction. Plasma exhibited the most potent inhibitory activity in these fluids. Fibrinogen was the most potent inhibitor of the plasma-derived proteins tested. These data suggest the possibility that the oral fluids may modulate the attachment of B. gingivalis to Gram-positive bacteria in periodontal pockets.

The presence of fucosyltransferases with different substrate specificity in human parotid saliva.

Using glycoproteins and milk oligosaccharides as substrate acceptors, we demonstrated at least two fucosyltransferases in human parotid saliva. One enzyme transferred L-fucose from GDP-fucose to the C-3 position of N-acetylglucosamine or glucose residue of oligosaccharide chains, and the other transferred to the C-4 position of N-acetylglucosamine residue of oligosaccharide chains.

Purification and characterization of galactosyltransferase in human parotid saliva.

The galactosyltransferase has been purified from human parotid saliva by ammonium sulfate precipitation (25-70% saturation), followed by repeated affinity chromatography on Sepharose-alpha-lactalbumin. The molecular weight of the enzyme was estimated to be approximately 56,000. The enzyme catalyzes the transfer of galactose from UDP-galactose to the exposed N-acetylglucosamine residues derived from glycoproteins, forming a Gal beta (1-4)GlcNAc linkage.

Effect of transferrin on the growth of Porphyromonas gingivalis.

This study describes the effect of transferrin as an iron source on the growth of Porphyromonas (formally Bacteroides) gingivalis. Bacterial growth was monitored spectrophotometrically. All strains of P. gingivalis tested grew well in medium containing transferrin. The growth of P. gingivalis depended not only on the concentration of transferrin, but also on the iron saturation level of the protein. However, growth was not stimulated with either the ferrous or ferric iron salts tested. The addition of dipyridyl to the medium containing transferrin suppressed the growth of P. gingivalis, which also did not show species-specificity for human transferrin. Transferrin-binding activity was found in P. gingivalis by solid-phase assay with peroxidase-conjugated human transferrin. These results suggest that P. gingivalis may be capable of utilizing transferrin as an iron source for growth in vivo.

Substrate specificity of fucosyltransferase purified from human parotid saliva.

The purified fucosyltransferase from human parotid saliva was shown to transfer fucose from GDP-fucose onto the oligosaccharide chains containing the Gal beta 1----3GlcNAc or Gal beta 1----4GlcNAc/Glc sequences. Competition studies between asialotransferrin and either lacto-N-fucopentaose 1 or 2'-fucosyllactose provided evidence that both the substrates competed for a common enzyme active site. These results suggest that the fucosyltransferase activities for the three acceptors may be catalyzed by the same enzyme.

Relationship between enzyme activities involved in oxygen metabolism and oxygen tolerance in black-pigmented Bacteroides.

In this study, the relationship between enzyme activities involved in oxygen metabolism and the degrees of oxygen tolerance in black-pigmented Bacteroides was investigated. All strains of Bacteroides tested possessed the activities of NADH oxidase and superoxide dismutase, whereas the activities of catalase and peroxidases were not detected in the cell-free extracts. There were relatively high correlations between oxygen tolerance and activity of either NADH oxidase or superoxide dismutase. The activity of superoxide dismutase showed a higher correlation with oxygen tolerance than with that of NADH oxidase. Among the species tested, Bacteroides gingivalis showed the highest activities of both the enzymes and was the most tolerant in the presence of oxygen. Furthermore, the activities of these two enzymes increased during aeration of the oxygen-tolerant strain Bacteroides gingivalis 381, but not in the oxygen-sensitive strain Bacteroides denticola ATCC 33185. These results suggest that superoxide dismutase and NADH oxidase might be important for protection of black-pigmented Bacteroides against the toxic effect of oxygen.