| Background Quorum sensing（QS）controls the development and maturity of various bacterial biofilms by regulating the communication about bacteria to bacteria.The N-acyl homoserine lactone-lactonase（AHL-Lactonase）,as a quorum quenching enzyme,disrupts the QS system via degrading QS-related signal molecules.At present,many studies have reported that AHL-lactonases effectively inhibit the formation of a variety of bacterial biofilms.Iso it is considered as a promising antibacterial strategy for prevention and treatment of bacterial infectious diseases without causing the problem of drug resistance.However,there are few studies in oral field.Therefore,this study aims to investigate the effects of AHL-Lactonase est816 on the formation of Aggregatibacter actinomycetemcomitans（A.actinomycetemcomitans）biofilm and release of virulence factors,and to explore the effects of est816 on the development of periodontitis in rats.Methods 6,12 and 24U/ml of est816 were co-cultured with A.actinomycetemcomitans suspension for 48 h to form mature biofilm,respectively.The effects of est816 on biofilm biomass and biofilm structure were detected by crystal violet staining,confocal laser scanning microscope（CLSM）and scanning electron microscopy（SEM）,respectively.The virulence factors of A.actinomycetemcomitans biofilm,such as the production of extracellular polysaccharide（EPS）,and the expression of cytolethal distending toxin A（cdt A）and leukotoxin A（lkt A）was detected by phenol sulfuric acid method,and realtime polymerase chain reaction（PCR）assays,respectively.The cytocompatibility of est816 on human gingival fibroblasts（HGF）was detected by CCK-8 and live/dead staining.Enzyme-linked immunosorbent assay（ELISA）was used to determine whether est816 could inhibit cell inflammation stimulated by A.actinomycetemcomitans biofilm supernatant.In vivo,periodontitis model in rats was established by ligation of silk thread,and SD rats were divided into control（no ligation+normal saline）,A.a（ligation+A.actinomycetemcomitans suspension）and est816+A.a（ligation+A.actinomycetemcomitans suspension+est816 regent）.To evaluate the degree of periodontitis inflammation in each group,maxillae were taken for micro-CT,HE and immunohistochemical staining at 1 and 2 months,respectively.16 S r RNA was examined to detect the difference in bacterial composition of gingival tissues in the ligated area of rats at 2 months.Results 1.Crystal violet staining,CLSM and SEM results showed that est816 significantly inhibited A.actinomycetemcomitans biofilm formation in a concentrationdependent manner（P<0.01）;2.est816 effectively suppressed virulence factors of A.actinomycetemcomitans biofilm,such as EPS,lkt A and cdt A（P<0.05）;3.CCK-8 and live/dead staining showed that 6 and 12U/ml est816 had no effect on the proliferation and viability of HGF;4.ELISA assay revealed that treatment HGFs with est816-pretreated biofilm supernatant alleviated the cell inflammation compared to stimulation with A.actinomycetemcomitans biofilm supernatant,and the expression of tumor necrosis factor-α and interleukin-6 decreased at 12 h（P<0.05）.5.Micro-CT and HE staining showed that the est816+A.a group significantly reduced alveolar bone absorption and inflammation severity of rats,and immunohistochemistry indicated that both the receptor activator of the NF-κB ligand and the expression of matrix metalloproteinase-9 related to bone resorption were reduced while osteoprotegerin increased in est816 group.16 S r RNA gene sequencing suggested that there was a certain difference in the ratio of bacterial composition between est816+A.a and A.a groups,but the difference in the bacterial composition of A.a group was more evident compared with the control group.Conclusion 1.AHL-lactonase est816 can dramatically attenuate A.actinomycetemcomitans biofilm formation and virulence factors release.2.est816 inhibits the cellular inflammatory response caused by A.actinomycetemcomitans biofilm with high biocompatibility.3.est816 can effectively control the development of periodontitis in vivo.In conclusion,this study highlights the therapeutic application of est816 in treating periodontitis and proposes a new strategy to address the problem of antibiotic resistance. |
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