Study On Kgp And Its Genotypes Of Porphyromonas Gingivalis In Subgingival Plaque In Puberty Gingivitis

OBJECTIVEStudies indicate that the incidence and severity of gingivitis rise in children during puberty. Their oral health may be damaged and their risk of developing periodontal diseases may be increased in the future. The composition of the flora in the oral cavity changes as the host grows and matures. Puberty appears to have a great effect on the composition of the periodontal flora. It is especially important to detect these bacteria during the period from healthy status to diseased status. Porphyromonas gingivalis ( P. gingivalis ) , a gram - negative anaerobic short rod, is one of the major causative periodontic bacterial that has been widely investigated. Investigators abroad reported that P. gingivalis was detected in 37% of children from 0 - to 18 - year - old subjects. However such studies in China are seldom seen.Cingipains, trypsin - like cysteine proteinases produced and secreted by P. gingivalis , appear to play an important role in the pathogenesis of periodontal disease. They are refered to gingipain R (Rgp) and gingipain K (Kgp) , depending on specificity for hydrolysis of either Arg - Xaa or Lys - Xaa peptide bonds, respectively. Gingipain K ( Kgp) is specifically involved in degradation of fibrinogen and fibrin, and thus may contribute to the bleeding tendency and persistent inflammation with P. gingivalis . Differences have been observed in the pathogenic potential of laboratory strains on the animal model or in vitro assay employed. Specific P. gingivalis genotypes are found to a greater extent in patients than in healthy subjects. Elucidation of possible association of genotypes with either disease or clinical healthy condition is important for understanding the pathogenic characteristics of bacteria.Investigations on major periodontic pathogens during puberty help to elucidate the natural history of periodontal diseases and to provide important evidences for screening susceptible crowd on the basis of genotypes and for prevention and treatment of them. Hie aims of the present study were as follows: to detect the presence of P. gingivalis in the subgingival plaque of puberty gingivitis and to probe into its relationship with gingival health using the more sensitive and specific method of 16S rRNA PCR;to assess the prevalence of specific kgp genotypes in puberty gingivitis as well as their possible association with disease severity;to detect the expression of Kgp by Western blot and its activity and to reveal the possible association of Kgp with puberty gingivitis.METHODS1. Study subjectsThe subjects of 14 - to 17 - year - old children in this study were separated into two groups, puberty gingivitis group and gingival healthy group, with 36 for each one. The subjects had received no periodontal therapy and any antibiotic therapy during the previous 3 months. No subjects with orthodontic and periodontic treatment were included. All subjects enrolled into the study signed the informed consent.2. Subgingival plaque sample collection and isolation of P. gingivalis Prior to the collection of subgingival plaque, clinical parameters includingGI, SBI, PD, PLI and CI were evaluated. After clinical examination, subgingival plaque samples were obtained with a sterile curette from the four first molars and anterior teeth. For the DNA isolation, the plaque samples were pooled in 1 ml of PBS in a microcentrifuge tube. For isolation of P. gingivalis , the samples were streaked on BHI blood agar plates. The plates were incubated at 37*0 in an anaerobic chamber for 5 to 7 days. Colonies of P. gingivalis were selected from their colony morphology and Gram's stain for identification. From each of these selected colonies a subculture was prepared. To confirm P. gingivalis in clinical isolates, P. gingivalis -specific 16S rRNA PCR was used.3. DNA isolationThe bacterial chromosomal DNA was prepared by phenol - chloroform extraction methods. The quantity of DNA was determined.4. Identification of P. gingivalis by 16S rRNA -based PCRA 16S rRNA - based PCR detection method was used to determine the prevalence of P. gingivalis in the subgingival plaque samples. The AVG of positive band in the 2% agarose gel electrophoresis was analyzed using the automatic gel documentation and image analyzer software.5. Identification of kgp genotypesPCR technique was used to amplify the region encoding the catalytic domain of kgp gene from the subgingival plaque DNA. The PCR products were se-quenced by use of ABI PRISM 310 Genetic Analyzer. The alignments of the sequence results were analyzed by BLAST, kgp genotypes were analyzed according to the digestion result with Msel.6. Preparation of protein samplesBacteria were grown in batches of 250 ml BHI base and harvested at the end of log - phase growth until reaching the same optical density ( OD^). The bacterial cultures were centrifuged (6,000 xg, 30min, 4t ) , and the supernatant (referred to as culture medium) was separated from pelleted cells. The latter were sonicated at 1,500 Hz for 10 min. Unbroken cells and large debris were removed by centrifugation (21,000 x g, 60min, 4*t! ) , and the cloudy supernatant was cell extract. Protein quantification was performed using Folin - Lowry method.7. Determination of Kgp activityAmidolytic activities of Kgp were assayed with N -p - Tosyl - Gly - pro -Lys 4 - nitroanilide - acetate salt. The absorbance oip - nitroaniline released from the substrate was measured spectrophotometrically at wavelength of 405 nm. The activity in individual fractions, calculated as percentage of the activity in the same fraction of strain W83, which was taken as 100%. The activities of Kgp in the samples were calculated.8. SDS -PAGE and Western blot analysisCulture fractions of P. gingivalis (culture medium and cell extract) were performed on 10% polyacrylamide gel. The gels were electrophoretic transferredonto membranes at a constant current of 50 V for 2 hours. Non - specific binding sites on the membranes were blocked over night in 5% bovine serum albumin and then immunoblotted. Hie blots were probed with primary antibodies against specific Kgp catalytic domain at a dilution of 1:500. Secondary antibody was goat anti - mouse IgC alkaline phosphate conjugate at a dilution of 1:2000. The AVC of positive blot was analyzed using the automatic gel documentation and image analyzer software.9. Statistical AnalysisThe data were statistically analyzed using SPSS11.5 software. The significance level was set at P < 0.05.RESULTS1. The prevalence of P. gingivalis in the puberty gingivitis group and gingi-val healthy group was 47.22% and 25.00% respectively ( x2 = 3. 85, P < 0. 05). The relative quantity of P. gingivalis in the two groups was 48.02% and 21.46% respectively ( P < 0.01). In all the P. gingivalis - positive subjects of puberty gingivitis group, there was positive correlation between the relative quantity of P. gingivalis and GI^ SBI and PD using Spearman correlation analysis ( P < 0.05). The GI and SBI index of the P. gingivalis -positive subjects were statistically higher than that of the P. gingivalis - negative subjects in the puberty gingivitis group using Rank sum test ( P < 0.01).2. The sequence analysis indicated the length of the PCR product of kgp catalytic domain was 792 bp. It was a fragment of kgp confirmed by BLAST with P. gingivalis W83 genome sequence.3. The restriction patterns allowed discrimination of two genotypes. All P. gingivalis positive subjects were subgingivally colonized by only one kgp genotype. The kgp - A genotype was digested in fragments of 102bp, 288bp and 402 bp, and kgp - B genotype was unrestricted with 792 bp. The prevalence of kgp - A genotype in puberty gingivitis group and gingival healthy group was 82.35% and 22.22% respectively. Fisher's exact test demonstrated that the distribution differences of genotypes between the two groups were statistically different ( P =0.021). There was no statistically significant difference in the clinical parameters in pubertal subjects harboring P. gingivalis oikgp -A genotype compared to that oikgp - B genotype( P > 0.05).4. Anti - Kgp N - terminal IgG subdomain immunoblottingCulture medium fractions from each strain revealed a reaction with a major band of 60 kDa in all samples. This band represents the catalytic domain of Kgp. An additional strong band (105 kDa) was visible too. While the cell extract fractions indicated positive bands of high molecular weight such as 120kDa, 105kD and 90kDa.5. Anti - Kgp caspase - like subdomain immunoblottingAt least 4 positive bands were seen in both fractions of 7 clinical isolates. They are 105, 90, 60, 45kDa. 32kDa and 27kDa bands occurred in some samples. 60kDa and 45kDa bands were not seen in three of the clinical isolates.6. There was positive correlation between the blotting intensity / activity of Kgp and the clinical parameters including GI, SBI, PD( P < 0.05).CONCLUSIONS1. P. gingivalis can colonize in the subgingival plaque in puberty subjects and its colonization is closely relevant to the status of gingival health.2. Restriction endonucleases analysis (REA) can divide P. gingivalis into two genotypes. Different kgp genotype may be relevant to the virulence of P. gingivalis . kgp - A genotype might be better adapted to environmetal challenges. Subjects with puberty gingivitis are mostly colonized by kgp -A genotype. It may be necessary to continue to monitor individuals who are positive for kgp - A genotype.3. There is a positive correlation between the Kgp blotting intensity/activity and clinical parameters such as GI^SBI and PD. Kgp is contributed to the path-ogenesis of puberty gingivitis.4. The Kgp in clinical isolates of P. gingivalis from puberty gingivitis is in complicated forms. Caspase - like molecules with low molecular weight may exist as intracellular functional protein molecules.