Study Of N-acetylglucosaminidase NagY1 And It's Effect To Actinobacillus Actinomycetemcomitans Biofilm

Biofilm is a height organized multicellular structure which consisted with bacterium and its secretory extracellular matrix and formed on the surface of object. Once the biofilm formed,the bacteria will become more resistant to antibiotic(100～1000 times higher than planktonic cells) and to the aggressive attack of immune system.The common existence of biofilm gives great trouble to the human beings life in many ways.According to the public announcement of NIH of America,more than 80%of bacterial infection concerned with biofilm.Because of the nearly ineffective of traditional antibacterials to the bacterial biofilm,thus,how to get rid of biofilm becomes a great challenge of human beings.Biofilm of dental plaque is the initiating agent of periodontal disease.At the stimulus of biofilm of dental plaque,the cytokine of periodontal tissue secretory plays an important role in the process of frontal resorption and periodontal histoclasia. However,the present majority study only focused on the cytotoxic of periodontal tissue induced by bacteriophankton.There's very few study at the role of bacterial biofilm to participate in periodontal disease.Extracellular polysaccharide is the major component of bacterial biofilm and determines the structure and function of biofilm. Actinobacillus actinomycetemcomitans(A.α) is the main pathogenic bacterium of invasive periodontitis.β-1,6-N-acetylglucosamine produced by A.αplays an important role in the structure and function of the biofilm.Therefore,this study wants to findβ-1,6-N-acetylglucosaminidase from ocean microorganisms and to study it's effects on A.αbiofilm.A marine bacterium strain QY403,which has a high yield ofβ-1,6-N- acetylglucosaminidase,is isolated from yellow sea mud sample using medium containingβ-1,6-N-acetylglucosamine as the sole carbon source.Strain QY403 is identified to belong to genus Pseudoalteromonas using molecular phylogenetic analysis based on the partial 16S rRNA gene sequence.Aβ-1,6-N-acetylglucosaminidase is purified from culture supernatants of strain QY403 after ammonium sulfate precipitation,ion exchange chromatography and gel filtration chromatography procedure.Molecular weight of this enzyme,which is named by NagY1,is 40.2 kDa.NagY1 is most active at 40℃and at pH6.0 and is stable below 40℃and over a range of pH5.0-7.2.Mg²⁺,Ca²⁺,Mn²⁺,Ba²⁺,Fe²⁺, Fe³⁺ and EDTA have the promotive effect on the enzymatic activity,while Cu²⁺ and Ni²⁺ have depressant effect,SDS has the deactivation effect.Substrate specificity analytic result of this enzyme showes that it only degradsβ-1,6-N-acetylglucosamine.On the foundation of the previous research,the effects of NagY1 on Actinobacillus actinomycetemcomitans(A.α) biofilm and cytokine production are investigated.At first,using the extra biofilm model of A.αto appraise the inhibition effect of different concentration of NagY1 on A.αbiofilm formation and detachment effect on the existed A.αbiofilm.The results show a prominent inhibition effect of NagY1 on A.αbiofilm formation(＞50%) and a detachment rate to the existed A.αbiofilm of 74.5%(P＜0.05).Then the effects of A.αbiofilm and A.αbiofilm degraded by NagY1 on the cytogenic of RANKL,IL-6,IL-1β,IL-8,IFN-γfrom the human periodontal ligament cells are investigated.The results show that both of biofilm cells and planktonic cells of A.αcan enhance the expression of IL-1β,IL-8 and IFN-γin human periodontal ligmament cells.The A.αbiofilm has more strong stimulation effect on cytogenic of RANKL,IL-6,IL-1β,IL-8 and IFN-γthan planktonic cells (P＜0.05).NagY1 can degrade A.αbiofilm and decrease comparatively cytokine mRNA expression.In summary,this study isolates one marine strain Pseudoalteromonas sp.QY403, and purified aβ-1,6-N-acetylglucosaminidase NagY1 from culture supernatants. NagY1 can effectively restrain A.αbiofilm formation and detach the existed biofilm of periodontal disease.Further more,NagY1 can remarkably decrease the cytogenic mRNA expression of human periodontal ligament cell.The results of this study can give rationale to the clinical use of NagY1,and open up a new approach to the prevention and cure of bacterium biofilm related periodontal disease.