Proteomic analysis of Enterococcus faecalis cell membrane proteins under alkaline stress conditions
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Date
2016
Authors
Cathro, Peter Robert
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Zilm, Peter Stephen
Kidd, Stephen Peter
Kidd, Stephen Peter
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Abstract
Background: Enterococcus faecalis is able to survive in a number of biological niches, which are often nutrient limited and in which the pH can vary greatly. Endodontic (root canal) treatment of a tooth with a severely inflamed, infected or necrotic pulp usually involves the chemo-mechanical debridement of the canal(s) using metal files, irrigants such as sodium hypochlorite and often inter-appointment medicaments such as calcium hydroxide (~pH 12.5 to 12.8) placed in the main root canal to help in the elimination of surviving bacteria. E. faecalis is commonly recovered from endodontic infections that have persisted following treatment with this highly alkaline medicament. The expression of the cell membrane proteins under alkaline conditions at a biologically relevant growth rate may increase our understanding of how E. faecalis can adapt and persist. Aims: 1. To determine the phenotypic changes of E. faecalis V583 when grown at a slow growth rate at pH 11. 2. To investigate and quantify cell membrane protein expression of E. faecalis V583, at pH 11 compared to pH 8, at an imposed growth rate using continuous culture. Methods: E. faecalis ATCC V583 was grown in a chemostat at pH 8 (control) and pH 11. Under each pH condition, the maximum growth rates were determined and an imposed growth rate of one-tenth the organism’s maximum growth rate (μrel) [rel subscript] was used for growth at pH 8 or 11. After steady state had been achieved, cells were harvested, lysed and membrane proteins were fractionated by ultracentrifugation, homogenisation in carbonate buffer, and membrane shaving. Following chymotrypsin digest (in the presence of RapiGest®) of the membrane fraction, heavy- or light-isotope-coding protein labels (ICPL) were added to samples from pH 8 or 11. Heavy-labelled (pH 11) and light-labelled (pH 8) samples were combined and the relative proportion of membrane proteins were identified using Liquid chromatography, electrospray ionisation (LC-ESI) mass spectrometry and MaxQuant analysis. The MaxQuant labelled ratios of membrane associated proteins were log₂ transformed, and the proteins that deviated by more than one standard deviation (SD) from the mean were considered to be up- or down-regulated. Results: The mean generation time at pH 8 was 1.16 hours and 7.7 hours at pH 11. One-tenth of the maximum growth rate (0.1 μrel) [rel subscript] was determined and set at 0.059 h⁻¹ for pH 8 and 0.009 h⁻¹ for pH 11. The extreme alkaline conditions produced co-aggregation of the cells into flocs (a variant of biofilm formation) with the appearance of an extracellular matrix. These observations are consistent with a shift towards spontaneous biofilm formation. Six proteins had a log2 H/L ratio (pH 11/pH 8) greater than one SD of the mean including: Polysaccharide biosynthesis family protein EF0669 (2SD), Glycosyl hydrolase, family 20 EF0114 (4SD), Glycerol uptake facilitator protein EF1927 (1SD), whilst five proteins had a log2 ratio one SD less of the mean: PTS system IIC component EF1838 (1D), PTS system IID component EF0456 (2SD), C4-dicarboxylate transporter EF0108 (1SD), PTS system mannose-specific IID component EF0022 (1SD). Conclusion: When cultured at an imposed slow growth rate, extreme alkaline conditions resulted in a reduced mean generation time and altered expression of several membrane proteins. Collectively these membrane proteins appear to be involved in the transition to biofilm formation seen at pH 11. It was hypothesised that the capsule observed at pH 11 protects the cell from destructive OH- ions whilst concentrating H+ ions and substrates required for the electrochemical gradient close to the cell membrane.
School/Discipline
School of Dentistry
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Dentistry, 2016.
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