Esterase from a cariogenic bacterium hydrolyzes dental resins

Abstract

Objectives: To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers.

Methods: Out of several putative esterases, an esterase encoded in an Open Reading Frame as SMU_118c (The National Center for Biotechnology Information, NCBI), was found to have true hydrolase activities. SMU_118c was cloned, expressed, purified and further characterized for its respective hydrolytic activity towards ester-containing nitrophenyl substrates and the universal resin monomers bis-phenyl-glycidyl-dimethacrylate (bisGMA) and triethyleneglycol dimethacrylate (TEGDMA) at neutral (7.0) or cariogenic (5.5) pH. Mass spectrometry (MS) was used to verify the expression of SMU_118c protein in S. mutans UA159.

Results: Similar to the whole cell activity of S. mutans, SMU_118c showed the highest affinity toward para-nitrophenyl acetate (pNPA) and para-nitrophenyl butyrate (pNPB) vs. ortho-nitrophenyl butyrate (oNPB) and butyrylthiocholine iodide (BTC) (p < 0.05). The esterase retained 60% of its activity after 21 days and hydrolyzed bisGMA at a higher rate than TEGDMA at both neutral and cariogenic pH (p < 0.001), similarly to the predominant human salivary esterase degradative activity. MS confirmed that SMU_118c is an intracellular protein in S. mutans UA159 and expressed under pathogenic (pH 5.5) growth conditions.

Significance: The similarity in the activity profile to the whole S. mutans bacterial cell, the stability over time at cariogenic pH, the preference to hydrolyze bisGMA and confirmed expression profile suggest that SMU_118c could be a significant contributor to the whole bacterial degradative activity of S. mutans toward the degradation of resin composites, adhesives and the restoration-tooth interface, potentially accelerating restoration's failure.

Statement of significance: The current study builds upon our highly-cited previous study by Bourbia et al., (JDR, 2013) that reported on that the cariogenic bacterium, S. mutans has esterase-like activities that enable the bacterium to degrade dental composites and adhesives. The current submission is the first to report on the isolation and characterization of the specific esterase activity (SMU_118c) from S. mutans that is a significant contributor to the whole bacterial degradative activity toward the hydrolysis of dental resins. This activity compromises the restoration-tooth interface, increases interfacial bacterial microleakage (Kermanshahi et al., JDR 2010), potentially contributing to the pathogenesis of recurrent caries around resin composite restorations. This represent a significant contribution to the field of biomaterials and their clinical performance.

Keywords: Biodegradation; Dental caries; Esterase; Hydrolysis; Resin monomers; Streptococcus mutans.

Fig. 1

(A) Nucleotide and deduced amino-acid sequences of SMU_118c (Preliminary identification of the active site is highlighted), and (B) SDS-PAGE of expression and purification of SMU_118c. An empty vector was used as a control to verify no expression of protein (not included in the final gel image).

Fig. 2

Activity profile of SMU_118c towards different nitro-phenyl substrates at pH 7.0 and pH 5.5 (n=3; data are reported as mean ± standard errors). *represents significant differences between the two pH values for each substrate (p < 0.05). Values with the different upper case letters denote statistically significant differences within pH 5.5 group (p < 0.05). Values with the different lower case letters indicate significant differences within pH 7.0 group (p < 0.05).

Fig. 3

The effect of different pH on the enzymatic activity of SMU_118c towards pNPB (A), and the relative effect of bisGMA and TEGDMA on SMU_118c activity at pH 7.0 (B) and pH 5.5 (C) following 21-day incubation (n=3; data are reported as mean ± standard errors). Values with the different lower letters indicate significant differences among different pH values (p < 0.05). *represents significant reduction after 21-day incubation (p < 0.05).

Fig. 4

SMU_118c catalyzed hydrolysis of bisGMA (A) or TEGDMA (B) at pH 7.0 and pH5.5 following up to 125-hour incubation. Results were normalized to buffer-only incubation condition (n=3; data are reported as mean ± standard errors).

Fig. 4

SMU_118c catalyzed hydrolysis of bisGMA (A) or TEGDMA (B) at pH 7.0 and pH5.5 following up to 125-hour incubation. Results were normalized to buffer-only incubation condition (n=3; data are reported as mean ± standard errors).

Fig. 5

The inhibitory effect of PMSF (0.5 mM) on the hydrolytic activities of SMU_118c towards (A) pNPB or (B) bisGMA (n=3; data are reported as mean ± standard errors). Values with the different lower case letters indicate significant differences among groups (p < 0.05).

Fig. 5

The inhibitory effect of PMSF (0.5 mM) on the hydrolytic activities of SMU_118c towards (A) pNPB or (B) bisGMA (n=3; data are reported as mean ± standard errors). Values with the different lower case letters indicate significant differences among groups (p < 0.05).