Comparing Genome Difference Between Porphyromonas Gingivalis Clinical Isolated Strains And Hypovirulent Strain ATCC 33277 With Array-based Comparative Genomic Hybridization Technique

ObjectivesThe purpose of the current study is to isolate Porphyromonas gingivalis ?P.gingivalis ? clinical strains of subgingival plaque from chronic periodontitis patients and compare the genomic differences with avirulent standard strain ATCC33277 with array-based Comparative Genomic Hybridization ?array-CGH? technique. We could discover the genomic DNA copy numbers varies and locate the abnormal fragments, which have great help in investigation of relationship between differential genes screened out and bacteria virulence. Utilizing the array-CGH technique may be a new tool in analysis of virulence genes, research of pathogenic mechanism of P. gingivalis, wide-scope filtering high-virulent strains infected patients, supporting the optimal target sites selection of gene vaccine and ultimately benefit patients with instructions about prevention and treatment of disease.Methods1. Subject population: 45 subjects with chronic periodontitis were included in the experimental group. Exclusion criteria were systemic disease, periodontal therapy and antibiotic administration within 6 months previously, smoking, pregnancy and nursing. We got the informed consent of all the subjects. The subgingival plaques of the most severe site and the health or with moderate gingivitis site were collected from each subject. Record the periodontal indexes of each sampling site,and all the exams were conducted by the same examiner to avoid bias.2. Sample collection and germiculture: subgingival plaque samples were obtained from sampling sites, transferred in transport buffer and anaerobic cultured in Brain Heart Infusion ?BHI? culture medium after dilution. The cultivate bacteria were collected after isolation, purified culture and enrichment. The genomic DNA of bacteria were extracted with genomic DNA extraction kit. The Polymerase Chain Reaction ?PCR? with specific primer of P. gingivalis was applied. Samples showed specific positive band would be recoreded and comfirmed by further sequencing anaylsis.3. Gene microarray: The independent designed microarray in previous study has covered the sequencing of 12 P. gingivalis strains, which have been published on the National Center for Biotechnology Information ?NCBI?.4. Microarray hybridization: The clinical isolated P. gingivalis strains and the minimally toxic strain P. gingivalis ATCC 33277 genomes were compared by array-CGH technique. The highly toxic strain P. gingivalis W83 were compared with ATCC 33277 as positive control and ATCC 33277 self-to-self hybridization as negative control. Genomes were enzyme digested, labeled,purified, 1:1 mixed and competitively hybridized to the microarray slides.5. Result analysis and PCR verification: The microarrays were scanned and signals of probs were assayed. The different genes were tested by the Basic Local Aligment Search Tool ?BLAST? to analyze the sequence alignment. PCR verify the accuracy of the result.6. Divergent genes analysis: A phylogenetic tree was created to analyse the homology of different P. gingivalis strains. The function of divergent genes between P. gingivalis strains and their relationship with bacteria virulence were analysed and revealed.Results1. We have successfully isolated 142 typical black single colonies from 90 subgingival plaques samples from 45 chronic periodontitis patients.2. 10 clinical isolated P. gingivalis strains were verified by specific PCR amplication and sequence alignment, 9 of which were successfully cultured,including 8 from severe sites with deep periodontal pocket and 1 from moderate gingivitis site. To ensure the genomic diversity, we only chose one strain from same sampling site, therefore only 6 clinical strains continue the further study.3. DNA genomes were competitively hybridized with P. gingivalis gene microarray. The negative control group, self-to-self hybridization of ATCC 33277, showed yellow fluorescence signals, while the hybridization of clinical isolated strains and ATCC 33277 mixtures showed different red and green mixed fluorescence signals. The results suggest that there are genetic diversity and heterogeneity between different P. gingivalis strains.4. Clinical strains showed higher similarity to toxic ones. There was barely significant difference of genomes between strains from mild gingivitis site and from severe periodontitis sites.5. The successive divergent DNA fragment deleted in clinical isolated strains PGN₀060 to PGN₀095, length in 34.5kb,are part of ATCC 33277 strain specific conjugative transposon CTnPg1-a, which are closely correlated with horizontal gene transfer. 102 genes copy number increase in more than 4 of clinical strains, all laid in genomes of high virulent strains W83 and TDC60,including two successive fragments, PG1473 to PG1490 and PGTDC60_RS04345 to PGTDC60_RS04405, length in 15.7kb and 17.4kb,which function closely to bacterial conjunction transfer and gene rearrangement The structure and composition of these fragments are coincide with Pathogenicity Islands ?PAIs? that might be virulence genes of P. gingivalis.6. Divergent genes were BLAST and verified by PCR, which demonstrated the accuracy and consistency of array-CGH technique.ConclusionsArray-CGH technique can be used to analyze virulence genes of P. gingivalis.The located divergent fragments lay the theory foundation for the further study of P. gingivalis pathogenicity mechanism and the selection of optimal target sites for the periodontitis gene vaccine design.