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Enterococcus faecalis is considered a predominant pathogen for persistent periapical infections and in addition is reportedly resistant to calcium hydroxide medication. The WalRK 2-component system of E. faecalis is essential for environmental adaptation, survival, and virulence. The goal of this study was to investigate the potential roles of walR in the regulation of biofilm aggregation, alkaline stress, and susceptibility to calcium hydroxide (CH) medication.Antisense walR RNA (aswalR) overexpression strains were constructed. Exopolysaccharide (EPS) production and bacterial viability of E. faecalis biofilms were evaluated by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to investigate the expressions of virulent factor genes. The proportion of viable bacteria and EPS production in dentin were assessed after CH medication.We showed that walR interference by aswalR RNA leads to a reduction in the dextran-dependent aggregation in E. faecalis biofilm. The overexpression of aswalR reduced the transcripts of the virulence genes and alkaline stress tolerance ability. Furthermore, the down-regulation of walR sensitized E. faecalis in infected canals to CH medication associated with inhibiting EPS synthesis.The data suggest a role for the walR regulator in the susceptibility to CH associated with dispelling the EPS matrix, which could be explored as a potential supplementary therapy for the management of root canal infection.