N -acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans

Abstract

Aims The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo . Results est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Quorum-quenching enzyme Est816 assisted antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in rats

“…Likewise, lactonase Est816, an antibiofilm agent, significantly increased the susceptibility of A. actinomycetemcomitans to various antibiotics. Based on our previous studies, we confirmed that enzyme Est816 had a significant effect on inhibiting biofilms ( Zhao et al., 2024 ). In this study, we investigated the synergistic effect of antibiotics and enzyme Est816 on biofilm inhibition, providing new support for further research on periodontal biofilm infections.…”

“…According to the previous study, expression and purification of Est816 was successfully prepared ( Fan et al., 2012 ). Based on our previous study findings ( Zhao et al., 2024 ), 12 U/mL of enzyme Est816 exerted a significant anti-biofilm effect against A. actinomycetemcomitans with minimal effect on bacterial growth and was used in this study.…”

“…A suspension of A. actinomycetemcomitans in mid-exponential phase was diluted to OD 600 = 0.1 (1.0 × 10 6 CFU/mL), and 500 µL were seeded onto round coverslips (14 mm diameter) on the bottom of 24-well plate, which was cultured with MTZ (sub- MIC or MIC), AMX (sub-MIC or MIC) and MINO (sub-MIC or MIC) alone or in combination with enzyme Est816 for 48 hours to form biofilms. Scanning electron microscopy (SEM) ( Zhao et al., 2024 ) was used to investigate the biofilm morphology. A. actinomycetemcomitans was cultured with MTZ (sub-MIC or MIC), AMX (sub-MIC or MIC) and MINO (sub-MIC or MIC) alone or in combination with the enzyme Est816 for 48 hours to form a biofilm, subsequently washed with PBS and fixed in 2.5% (vol/vol) cold (4°C) glutaraldehyde overnight.…”