Fimbriae Proteins ; genetics ; Genotype ; Humans ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Virulence

OBJECTIVETo determine the sequence of the active peptide derived from recombinant hemagglutinin (rHA-2) of Porphyromonas gingivalis (Pg).

METHODSThe HA-2 gene from PgATCC33277 was cloned, expressed in Escherichia coli (Ec) BL21 (DE3), and purified. The purified recombinant protein was evaluated for its ability to bind hemin-linked agarose. The active peptide was subjected to endoproteinase-mediated sequence analysis.

RESULTSThe protein expressed in Ec BL21 (DE3) was identified as PgHA-2 by plasmid sequence analysis, Western blotting, and mass spectrometry. The recombinant protein was confirmed functional by its ability to bind hemin. The sequence of the active peptide of rHA-2 was determined to be DHYAVMISKTGTNAG.

CONCLUSIONSThe availability of sequence of the active peptide of rHA-2 provides a foundation for the development of immunoprophylactic and therapeutic agents against this human pathogen.

Bacterial Proteins ; chemistry ; genetics ; Hemagglutinins ; chemistry ; genetics ; Porphyromonas gingivalis ; genetics ; Sequence Analysis, Protein

OBJECTIVETo construct and identify the Porphyromonas gingivalis(Pg)ATCC33277 hemagglutinin-2(HA-2)-deficient mutant.

METHODSThe genomic DNA of Pg was isolated from PgATCC33277. The up/down stream genes of HA-2-HA(u), HA(l) were amplified by PCR, and inserted into pSY118 separately which contains a 2.1 kb antibiotic resistance ermF-ermAM cassette. The resultant recombinant plasmid-pSY118-HA was linearized as the gene targating fragment HA-ermF-ermAM and used in the electroporation of PgATCC33277. The Pg HA-2-deficient mutant was screened by allelic exchange. The test of aggregation of red blood cells was used to investigate the function change between PgHA2-deficient mutant and the wild type of PgATCC33277.

RESULTSThe PgHA-2-deficient mutant was identified by PCR. The ability of Pg HA-2-deficient mutant to aggregate red blood cell was significantly decreased compared with the wild type.

CONCLUSIONSHA-2-deficient mutant of Pg ATCC33277 was constructed successfully, which lays a foundation for further study of its biological function.

Alleles ; Erythrocyte Aggregation ; Hemagglutinins ; genetics ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; genetics

OBJECTIVETo identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277.

METHODSUsing suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically.

RESULTSSubtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.

CONCLUSIONSComparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.

Genes, Bacterial ; Genome, Bacterial ; Nucleic Acid Hybridization ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Sequence Analysis, DNA ; Virulence ; genetics

OBJECTIVEThis study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.

METHODSEukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.

RESULTSIt proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.

CONCLUSIONThe eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

Animals ; Bacterial Proteins ; genetics ; COS Cells ; Cercopithecus aethiops ; Cysteine Endopeptidases ; genetics ; Genetic Vectors ; Plasmids ; Porphyromonas gingivalis ; genetics ; Transfection

Objective: To detect and analyze the characteristics of oral microbiota in species composition, function and metabolism among caries, periodontitis and oral healthy individuals, hunting for the microbiome-derived biomarkers with specificity and sensitivity to estimate the occurrence of these two diseases. Methods: Saliva samples were collected from 10 patients with high caries risk [decayed-missing-filled teeth (DMFT)≥6, HC group] in Department of Endodontics, 10 patients with periodontitis of grade Ⅱ A-Ⅲ C (PG group) in Department of Periodontology and 10 oral healthy individuals (HH group) from School of Stomatology, The Fourth Military Medical University during from March 2022 to June 2022. A baseline examination was conducted on all participants, including their oral conditions of caries and periodontal health. Metagenomic sequencing (Illumina PE150 platform) and liquid chromatography-mass spectrometry were used to detect microorganisms and their metabolites in the samples respectively. The sequencing data were analyzed to obtain the information of microbial taxonomic composition, functional genes and metabolites in each group of samples. The basic oral conditions and saliva samples of subjects in each group were evaluated and collected by the same professional endodontist. Results: There were no significant difference in baseline characteristics such as age and sex among the subjects in each group (P>0.05). DMFT in HC group (9.0±1.7) was significantly higher than that in HH group (0) and PG group (0) (F=243.00, P<0.001). Sequencing data analysis showed that the taxonomic compositions of salivary microbiota in each group were mainly Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria at the phylum level, and Streptococcus, Neisseria, Rothia, Prevotella at the genus level. Differential analysis showed that, compared with the HH group, HC group and PG group had significant differences in taxonomic composition (P<0.05), and the most significant among them was Prevotella. At the species level, Prevotella pallens was the most significant change in HC group, and Porphyromonas gingivalis in PG group. Metabolite analysis showed that there were significant differences in metabolites between HC group and PG group. The results showed that, compared with the HH group, the most significant metabolite change was 3-hydroxy-1, 5-diphenylpentan-1-one in HC group (P=0.001) and N1 acetylspermine in PG group (P=0.002) respectively. Compared with the PG group, the metabolite of HC group with the most significant difference is D-glucosamine 6-phosphate (P=0.006). The metabolism gene function analysis showed that, the enrichment of carbohydrate metabolism related genes was highest in HC group, followed with HH group, and it was lowest in PG group. In addition, compared with the HH group, the abundance of functional genes related to glucose metabolism, such as ABC transporter and phosphotransferase system, were significantly decreased in PG group (P<0.05), but significantly increased in HC group (P<0.05). Conclusions: There is a significant correlation between the alternation of carbohydrate metabolism of salivary microbiota with the occurrence of caries and periodontitis. In the future, Prevotella pallens and 3-hydroxy-1, 5-diphenylpentan-1-one may be the potential biomarkers of caries; while Porphyromonas gingivalis and N1 acetylspermine work in the predictions of periodontitis.
Humans ; Saliva/microbiology* ; Dental Caries Susceptibility ; Periodontitis/microbiology* ; Microbiota/genetics* ; Porphyromonas gingivalis/genetics* ; RNA, Ribosomal, 16S/genetics*

OBJECTIVETo investigate the mechanism of monocyte chemoattractant protein-1 (MCP-1) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.

METHODSPg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MCP-1 expression in HGF. MCP-1 mRNA levels of HGF were determined by real-time RT-PCR and MCP-1 protein levels in culture supernatant by ELISA at different time intervals (1 h, 3h, 6h and 12h) following continuous co-culture of bacteria with HGF.

RESULTSMCP-1 mRNA and protein levels were both up-regulated when HGF co-cultured with different Pg fimA genotypes. Type II was stronger than other fimA genotypes, HGF expressed significantly great amount of MCP-1 mRNA [(25.75 +/- 3.12)-(326.69 +/- 35.35)] and protein [(178.20 +/- 46.20)-(443.46 82.19) ng/L] for different time periods; While Type III was weaker than other fimA genotypes, and the level of MCP-1 mRNA was [ (4.16 +/- 0.82)-(94.17 +/- 18.56)] and protein [(86.95 +/- 23.90)-(264.01 +/- 28.59) ng/L](P < 0.05).

CONCLUSIONSfimA genotypes of Pg are related with the inductions of MCP-1, which might indicate fimA genotype is associated with pathogenesis of Pg.

Cells, Cultured ; Chemokine CCL2 ; metabolism ; Fibroblasts ; metabolism ; Fimbriae Proteins ; genetics ; Genotype ; Gingiva ; cytology ; microbiology ; Humans ; Porphyromonas gingivalis ; genetics

OBJECTIVETo investigate the distribution of fimA genotype of P. gingivalis in chronic periodontitis patients.

METHODSSubgingival plaque samples were collected from 101 chronic periodontitis patients. P. gingivalis was detected by both culture method and P. gingivalis 16S rRNA PCR. fimA type-specific primer were designed, and the distribution of fimA genotype of P. gingivalis in periodontitis patients were detected by PCR.

RESULTSThe detective ratio of P. gingivalis was 88.1%. Among them, a single fimA genotype was detected in most subgingival plaque samples (65.1%), and the distribution of five fimA genotypes among P. gingivalis positive patients was as follows: type I, 24.7%; type II, 43.8%; type III, 15.7%; type IV, 40.4%; type V, 3.4%; respectively.

CONCLUSIONP. gingivalis with various fimA genotypes were present in subgingival plaque samples from chronic periodontitis patients, and P. gingivalis with type II fimA and IV fimA were more predominant in chronic periodontitis patients, and they may be associated with the development of periodontitis.

Adult ; Chronic Periodontitis ; microbiology ; Dental Plaque ; Female ; Fimbriae Proteins ; genetics ; Genotype ; Humans ; Male ; Periodontitis ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; genetics

OBJECTIVETo assess the prevalence of specific kgp genotypes in puberty gingivitis and investigate their possible association with disease severity.

METHODSSubgingival plaque samples were collected from 72 pubertal children aged from 14 to 17 years, which were divided into two groups, gingivitis group and healthy (control) group. Clinical parameters were recorded beforehand. PCR technique was used to amplify the region encoding the catalytic domain of gingipain K (KGP). The PCR products were digested with restriction enzymes Mse I.

RESULTSThe kgp-A genotype was digested in fragments of 102 bp, 288 bp and 402 bp, and kgp-B genotype was unrestricted with 792 bp. Virulent strain P. gingivalis W83 was manifested by kgp-A genotype while a virulent strain P. gingivalis ATCC 33277 was manifested by kgp-B genotype. All P. gingivalis positive subjects were subgingivally colonized by only one kgp genotype. The prevalence of kgp-A genotype in puberty gingivitis group and gingival healthy group was 79.0% and 22.2% respectively. The distribution differences of genotypes between the two groups were statistically different (P = 0.028). There was no statistically significant difference in the clinical parameters between pubertal subjects harboring P. gingivalis of kgp-A genotype and kgp-B genotype (P > 0.05).

CONCLUSIONSMost subjects with puberty gingivitis were harbored by the same kgp genotype as that of virulent strain P. gingivalis W83. It may be necessary to continue to monitor individuals who are positive for P. gingivalis of kgp-A genotype since their risk of developing periodontal diseases may be increased in the future.

Adolescent ; Case-Control Studies ; Dental Plaque ; microbiology ; Female ; Genotype ; Gingivitis ; microbiology ; Humans ; Male ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; genetics

OBJECTIVE: To describe the submucosal microbial profiles of peri-implantitis and healthy implants, and to explore bacteria that might be correlated with clinical parameters. METHODS: In the present cross-sectional study, 49 patients were recruited. Each patient contributed with one implant, submucosal biofilms were collected from 20 healthy implants and 29 implants with peri-implantitis. DNA was extracted and bacterial 16S ribosomal RNA (16S rRNA) genes were amplified. Submucosal biofilms were analyzed using 16S rRNA sequencing at Illumina MiSeq platform. Differences between the groups were determined by analyzing α diversity, microbial component and microbial structure. The potential correlation between the bacteria with pocket probing depth (PPD) of peri-implant calculated by Spearman correlation analysis. RESULTS: The α diversity of submucosal microbial of health group was significantly lower than that in peri-implantitis group (Chao1 index: 236.85±66.13 vs. 150.54±57.43, P < 0.001; Shannon index: 3.42±0.48 vs. 3.02±0.65, P=0.032). Principal coordinated analysis showed that the submucosal microbial structure had significant difference between healthy and peri-implantitis groups [R²=0.243, P=0.001, analysis of similarities (ANOSIM)]. Compared with healthy implants, relative abundance of periodontal pathogens were higher in peri-implantitis, including members of the red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and some members of orange complex (Precotella intermedia, Eubacterium nodatum, Parvimonas micra), as well as some new periodontal pathogens, such as Fillifactor alocis, Fretibacterium fastidiosum, Desulfobulbus sp._HMT_041, and Porphyromonas endodontalis. Spearman correlation analysis revealed that the relative abundance of Treponema denticola (r=0.686, P < 0.001), Tannerella forsythia (r=0.675, P < 0.001), Fretibacterium sp. (r=0.671, P < 0.001), Desulfobulbus sp._HMT_041 (r=0.664, P < 0.001), Filifactor alocis (r=0.642, P < 0.001), Fretibacterium fastidiosum (r=0.604, P < 0.001), Porphyromonas gingivalis (r=0.597, P < 0.001), Porphyromonas endodontalis (r=0.573, P < 0.001) were positive correlated with PPD. While the relative abundance of Rothia aeria (r=-0.615, P < 0.001) showed negatively correlation with PPD. CONCLUSION Marked differences were observed in the microbial profiles of healthy implants and peri-implantitis. The members of red and orange complex as well as some new periodontal pathogens seem to play an important role in peri-implant disease. Compared with healthy implants, the submucosal microbial of peri-implantitis were characterized by high species richness and diversity.
Humans ; Peri-Implantitis/microbiology* ; Cross-Sectional Studies ; RNA, Ribosomal, 16S/genetics* ; Bacterial Load ; Porphyromonas gingivalis ; Dental Implants