|dc.description.abstract||Dental root canal treatment was first attempted in 1728 (Hasegawa 1983), and is routinely performed all over the world, despite a significant failure rate. The primary cause of failure is bacterial infection, and the most commonly associated bacterium from secondary root canal infections is Enterococcus faecalis with isolation rate ranging from 30 to 70 %.
E. faecalis produces biofilms and tolerates the high alkalinity of calcium hydroxide; the most commonly used intracanal medication. When conditions inside the root canal become extreme (low nutrients and high pH), E. faecalis is selected leading to inflammation of the periapical tissues and consequently treatment failure.
One proposed mechanism for the adaptation of E. faecalis to high pH is the involvement of two membrane proteins that are upregulated under conditions of nutrient deprivation and elevated alkalinity; specifically glycosyl hydrolase (Ef0114) and glycerol facilitator membrane protein (GlpF) (Ef1927).
The overall aim of this study was to assess the involvement of glycosyl hydrolase and GlpF in alkaline tolerance and biofilm formation by E. faecalis.
The specific aims were:
(i) to compare alkaline tolerance among root canals isolates of E. faecalis and non-root canal isolates in order to determine if the conditions within the treated root canal select strains better able to adapt and survive.
(ii) To investigate the involvement of the glycosyl hydrolase and GlpF in alkaline tolerance and biofilm formation. The up-regulation of these membrane proteins could be related to a generalized stress response rather than a specific reaction to the alkaline condition, and targeting these proteins could offer alternative treatment strategies.
The study revealed considerable variability in alkaline tolerance among strains of E. faecalis, and the root canal isolates were no more tolerant than non-root canal strains. GlpF is a highly conserved membrane protein with minimal variation among E. faecalis strains. Whereas, the glycosyl hydrolase showed amino acid variations but with no correlation to alkaline tolerance.
Biofilm formation was variable among strains of E. faecalis; however it was highly influenced by the type of carbohydrate available and the pH. Thicker biofilms were formed in the presence of glucose compared to glycerol, and at pH 8 compared to pH 11. In addition, the metabolic activity of the biofilm bacteria was significantly higher when the biofilm was originally formed in glycerol compared to glucose. Blocking of the glycosyl hydrolase with PUGNAc (analogue of N-acetyl glucosamine) significantly decreased the biofilm biomass formed in glucose and the metabolic activity of biofilm formed in glycerol.
Glycerol metabolism contributed to the alkaline tolerance of E. faecalis by accelerating the growth of the least tolerant strain at pH 11, and by increasing the metabolic activity of the biofilm bacteria subjected to high pH.
Genetic modification of E. faecalis was challenging. Various approaches were therefore attempted to derive mutants of the two genes of interest including deletion mutation and transposon mutagenesis. Ef0114 (encoding glycosyl hydrolase) was successfully deleted, while potential mutants of Ef1927 (encoding GlpF) reverted to the wild type, strongly suggesting that GlpF is essential for survival of E. faecalis.
Screening of a library of 5000 transposon derivatives did not result in any alkaline-sensitive mutants. However, two transposon mutants of each of the targeted genes were identified in a previously sequenced transposon library providing opportunity for investigating their involvement in alkaline tolerance and biofilm formation.
Both the glycosyl hydrolase deletion mutant and the GlpF transposon mutants showed minimal difference in tolerance from the wild-type strain. On the other hand, a GlpF over expressing strain exhibited accelerated growth at pH 11 compared to the wild-type, and GlpF downregulation in a Ers (Enterococcus regulator of survival) mutant decreased the metabolic activity of the biofilm, suggesting a contribution to alkaline tolerance.
Application of glycosyl hydrolase inhibitor (PUGNAc) led to the conclusion that the contribution of glycosyl hydrolase to alkaline tolerance is in part due to metabolism of complex glycoproteins that provide energy and results in acidification of the alkaline environment. However, this effect is likely a contributor rather than the principal mechanism of surviving high pH.
Of note, Auphen (gold III compound), a recognised inhibitor of mammalian GlpF, demonstrated an effective antimicrobial action against E. faecalis, providing encouraging evidence that GlpF constitutes an accessible target for effective antimicrobial treatment.||