Diversity Of Porphromonas Gingivalis Outer Membrane Protein On Inducing Different Cells To Secrete Inflammatory Cytokines

Chronic periodontitis(CP) is one of the common oral infectious diseases in human beings which is affected by multiple factors.Recently researches have demonstrated that periodontitis is elicited by suppression of periodontal immune defenses,colonization and overgrowth of periodontal bacterial pathogens which produce an array of virulence factors,release of inflammatory cytokines and chemokines by host cells,initiation of cytotoxic or immunopathological change, and subsequently periodontal tissue breakdown,at last odontoptosis happen.It is a major cause for teeth loss in the adult population.Being one of the well confirmed putative periodontal pathogens, Porphyromonas gingivalis(Pg),a gram-negative,oral anaerobic black-pigmented bacterial species,expresses a number of potential virulent factors that enable it to evade innate immmune defense system and destroy host cells,including lipopolysaccharides(LPS),fimbriae,collagenase,gingipain,outer membrane protein,hemagglutinin and a superoxide dismutase,etc,but pathogenic mechananism of the microb remains not to be completely understood.It has generally accepted that bacterial fimbia plays a important role in its pathogenic process.P.gingivalis was found to have fimbria periodontal epithelial cells.Porphyromonas gingivalis outer membrane protein A(PgmA),found by Hongo et al.in 1999,possesses the function which is related to fimbrial morphogenesis.Synthesis of P.gingivalis fimbrial protein would be blocked if pgmA gene deletion.Since high incidence of periodontitis is present,vaccination of multivalence genetic engineering vaccine prepared with suface protein antigens of dominant periodontitis-causeing bacteria was considered as a possible way to prevent and control the disease.Therefore,we clone pgmA gene from P.gingivalis genome DNA,constructed expression system of the pgmA gene,established methods to extract and purify the expressed products.But the capability of rpmA in activating many kinds of target cells in periodontal tissues,such as immunologic reaction cells(macrophage and lymphocyte),neutrophils which having defensive function,major cells of periodontal tissues(fibroblast),osteoblasts and osteoclast, etc.and its role in making these cells synthesize and secret various kinds of cytokines and inflammatory mediators for the damage of periodontal tissues, remains controversial.Therefore,it is necessary to make further discuss to this study.We also hope to perform some preparative works for thoroughly studying of the generation and development of periodontitis and improving the levels of periodontitis prevention and treatment.Objective:To determine the capability and diversity of Porphyromonas gingivalis outer membrane protein A(PgmA)inducing human oral epithelial ceil(KB cells),human gingival fibroblasts(HGF-1 cells) and human mononuclear macrophagocytes cell (THP-1 cells) to secrete inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-8.Method:1,Genome DNA of P.gingivalis strains ATCC 33277 and 47A-1 were prepared by using routine phenol-chloroform method.Entire pgmA gene from the DNA were amplified by high fidelity PCR.The nucleotide sequences of the target DNA amplification fragments were sequenced after T-A cloning.Plasmid pET32a was used to construct prokaryotic expression vectors of the cloned pgmA gene.The target recombinant proteins rPgmA were expressed in host cell of E.coliBL21DE3 induced by IPTG with different dosages.Ni-NTA affinity chromatography was used to extract and purify rPgmA.Westen blots were applied to determine immunoreactivity and antigenicity of rPgmA by using rabbit antisera against whole cell of P.gingivalis and the recombinant proteins as the first antibodies,respectively.2,By using different dosages rPgmA stimulate KB,HGF-1,THP-1 for different duration time,we extracted their supernatants and then used quantitative ELISA kits to test the changes of TNF-α,IL-1β,IL-6 and IL-8 levels.Results:1,PCR:Target amplification fragments of pgmA genes from DNAs of the strains were obtained and the sizes of the fragment was approximately 1473bp,respectively.2,Analysis results of the nucleotide and putative amino acid sequences:Nucleotid sequences of the cloned pgmA genes from P.gingivlis strain ATCC 33277 were completely same as those from P.gingivalis strain 47-1,respectively.In comparison with the corresponding nucleotide and putative amino acid sequences from GeneBank,similarities were 98.90%and 99.17%for the cloned pgmA gene,respectively.3,Exraction,purification and identification of the expression products:The results of SDS-PAGE demonstrated that IPTG at different dosages of 1.0,0.5,0.1 and 0.05 mmol/L could efficiently induce expression of rpgmA.Inclusions were mainly presented forms for the expressed proteins.Outputs of rpgmA were approximately 50%of the total bacterial proteins,respectively.4,Results of Western blot detection:Both of rPgmA could combine with the rabbit antiserum against whole cell of P.gingivalis and induce rabbits to produce specific antibodies,respectively.5,Neither Pg-LPS nor E-LPS could induce KB cells to secrete the cytokines mentioned above.6,The rPgmA could induce HGF-1 cells to secrete TNF-αand IL-1βlevels which were remarkably increased with a perk model(P＜0.01),while a continuous enhancement model of the two eytokines for THP-1 cells was presented(P＜0.01).7,The rPgmA had effects to promote HGF-1 cells or THP-1 cells to continuously heighten the secretion of IL-6(P＜0.01).8,rPgmA were able to induce increase of IL-8 secretion in THP-1 cells (P＜0.01),The rPgmA showed the similar effect in HGF-1 cells(P＜0.01).Conclutions:1,The rPgmA has powerful activity to promote its target cells to increase their secretion of several inflammatory cytokines,which causing initial local inflammation.2,KB cell can not used as the target cells to determine inflammation causing effect of rPgmA.