Effects of ClpP protease on biofilm formation of Enterococcus faecalis

Abstract Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms. Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student’s t-test or one-way analysis of variance (ANOVA). Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis. Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.摘要：粪肠球菌（Enterococcus faecalis，以下简称E. faecalis）是引发难治性根尖周炎与医院感染的主要病原菌之一，其在生物膜中的致病力显著增强。 研究目的：已有研究表明，酪蛋白水解蛋白酶P（caseinolytic protease P，以下简称ClpP）参与生物膜形成过程。然而截至目前，鲜有研究探讨ClpP在粪肠球菌存活及促进生物膜形成中的作用。因此本研究旨在分析ClpP对粪肠球菌生物膜形成的影响。 研究方法：本研究采用同源重组技术（homologous recombination）构建粪肠球菌ATCC 29212的clpP基因缺失株与互补株。通过活菌菌落计数法（viable colony counting method）分析粪肠球菌的生长模式；采用结晶紫染色（crystal violet staining，以下简称CV）与共聚焦激光扫描显微镜（confocal scanning laser microscopy，以下简称CLSM）表征生物膜的形成量，借助扫描电子显微镜（scanning electron microscopy，以下简称SEM）观察生物膜的微观结构。采用t检验（Student’s t-test）或单因素方差分析（one-way analysis of variance，以下简称ANOVA）对数据进行统计学分析。 研究结果：结果显示，clpP基因缺失株的生长模式发生改变，且其胞外多糖基质含量减少，相较于标准菌株，该缺失株的生物膜形成能力显著下降。此外，实验证实ClpP可增强粪肠球菌的生物膜形成能力。 研究结论：本研究证实ClpP可提升粪肠球菌的生物膜形成能力，并强调了ClpP作为抗粪肠球菌潜在药物靶点的重要性。