Evidence for Proline Utilization by Oral Bacterial Biofilms Grown in Saliva

Abstract

Within the mouth bacteria are starved of saccharides as their main nutrient source between meals and it is unclear what drives their metabolism. Previously oral in vitro biofilms grown in saliva have shown proteolytic degradation of salivary proteins and increased extracellular proline. Although arginine and glucose have been shown before to have an effect on oral biofilm growth and activity, there is limited evidence for proline. Nuclear magnetic resonance (NMR) spectroscopy was used to identify extracellular metabolites produced by bacteria in oral biofilms grown on hydroxyapatite discs. Biofilms were inoculated with stimulated whole mouth saliva and then grown for 7 days using sterilized stimulated whole mouth saliva supplemented with proline, arginine or glucose as a growth-medium. Overall proline had a beneficial effect on biofilm growth—with significantly fewer dead bacteria present by biomass and surface area of the biofilms (p < 0.05). Where arginine and glucose significantly increased and decreased pH, respectively, the pH of proline supplemented biofilms remained neutral at pH 7.3–7.5. SDS-polyacrylamide gel electrophoresis of the spent saliva from proline and arginine supplemented biofilms showed inhibition of salivary protein degradation of immature biofilms. NMR analysis of the spent saliva revealed that proline supplemented biofilms were metabolically similar to unsupplemented biofilms, but these biofilms actively metabolized proline to 5-aminopentanoate, butyrate and propionate, and actively utilized glycine. This study shows that in a nutrient limited environment, proline has a beneficial effect on in vitro oral biofilms grown from a saliva inoculum.

Novel bacterial proteolytic and metabolic activity associated with dental erosion-induced oral dysbiosis

“…The identification of several bacterial proteases (msrAB, vanY and ppdC, see Table 1) over-expressed in the erosion group also suggested proteolytic activity in saliva from subjects with erosion. We have shown previously that the presence of proline, glycine, 5-aminopentanoate, butyrate, acetate, and propionate are all metabolic markers of increased bacterial proteolysis [19,40]. This study may also suggest that PIP and ZAG degradation are associated with the presence of Prevotella in the oral cavity which may be a candidate species to degrade these proteins, as this genus was seen in higher abundance in PIP and/or ZAG degraders (not significant, data not shown).…”

Metataxonomic and metabolomic evidence of biofilm homeostasis disruption related to caries: An in vitro study

“…Proline has recently been shown to play a role in pathogen and host interactions inside and outside the oral cavity by modulating cell signaling and osmotic stress and acting as an antibacterial molecule or prebiotic (Christgen & Becker, 2019;Cleaver et al, 2019Cleaver et al, , 2020. Slomka et al (2017) investigated a proline-containing prebiotic candidate based on methionine-proline, which after just three days of exposure shifted the composition of an oral biofilm model by reducing the pathogenic species to a predominantly beneficial species (Slomka et al, 2017(Slomka et al, , 2018.…”

“…Proline has recently been shown to play a role in pathogen and host interactions inside and outside the oral cavity by modulating cell signaling and osmotic stress and acting as an antibacterial molecule or prebiotic (Christgen & Becker, 2019 ; Cleaver et al., 2019 , 2020 ). Slomka et al.…”

How to study biofilms: technological advancements in clinical biofilm research

“…Although CLSM uses fluorescence to visualise samples, it is different from fluorescence microscopy due to the pinpoint laser which improves resolution and the scanning of the laser through the sample on the x, y and z planes which provides z -stack images which can be reconstructed using open-source software such as COMSTAT2 ( Vorregaard, 2008 ; Samarian et al., 2014 ) and BiofilmQ ( Hartmann et al., 2021 ) . The information obtained from z -stack image analysis can provide information on biofilm depth, biomass, and surface area which is useful to compare biofilm characteristics when exposed to different molecules, along with live/dead staining ( Figures 2A, B ) which can show the distribution of bacterial viability within the biofilm ( Bridier et al., 2010 ; Morris et al., 2020 ; Cleaver et al., 2021 ). Specific dyes and probes can be used, such as FM 1-43, fluorescent in situ hybridisation (FISH) probes ( Thurnheer et al., 2004 ; Malic et al., 2009 ; Lebeer et al., 2011 ), and carboxy-SNARF ( Corsini et al., 2021 ) for the monitoring of pH within the biofilm.…”