IntroductionSalivary mucin MUC5B has been suggested to support eubiosis in early oral biofilms by regulating the attachment of commensals, while downregulating dysbiotic activities related to dental caries development, such as microbial carbohydrate transport and metabolism.MethodsTo investigate how the metabolism of glucose, a potential driver for dental caries, in early mono- and dual-species biofilms of oral Actinomyces naeslundii and Streptococcus gordonii clinical isolates was affected by the presence of the complex salivary mucin MUC5B, this study employed nuclear magnetic resonance (NMR)-based metabolomics with the interpretation of network integration.Results and discussionMUC5B reduced early attachment in the presence of glucose compared with uncoated surfaces but maintained even species distribution. This suggests that MUC5B may represent an innate mechanism to regulate biofilm eubiosis by supporting early coadhesion while regulating total biomass. All annotated metabolites were intermediates in either carbohydrate metabolism, pyruvate conversion, or amino acid metabolism, which was not unexpected in biofilm glucose metabolomes from two saccharolytic species since pyruvate conversion represents a junction point between glycolysis and amino acid metabolic chains. The 10 metabolites present in all early biofilms represent a core metabolome shared by A. naeslundii and S. gordonii. Such core metabolomes can be used to detect deviations in future studies. Significant differences in metabolite abundance elicited by the presence of MUC5B were also detected. In early biofilms where they were each present, pyruvate, ethanol, and metabolite 134 were present in significantly higher abundance in the presence of 25% MUC5B with 20 mM glucose (MUC5B + G) compared with a physiologic buffer with 20 mM glucose (PBS + G), while metabolites 84, 97, and sarcosine were present at significantly lower abundance. Metabolite 72 was unique to biofilms grown in MUC5B + G, and eight unannotated metabolites were unique to biofilms grown in PBS + G. A pathway enrichment analysis of the metabolites that were differently expressed in early A. naeslundii, S. gordonii, and dual-species biofilms grown with 20 mM glucose with or without MUC5B showed that pyruvate metabolism was significantly over-represented. Studying the metabolic interactions between commensal members of oral biofilms and modulatory effects of host factors such as glycoproteins in saliva during the metabolism of substrates that are potential drivers of dysbiosis, such as glucose, is essential to understand the roles of oral microbial ecosystems in oral health and disease.

Background Mucin degradation is essential for understanding oral microbial adaptation, yet the enzymes involved remain incompletely understood. Herein, we have characterised two mucin-degrading proteases, MdpS and MdpS2, from the oral commensal Streptococcus oralis.Materials and methods MdpS2 was characterised using physicochemical assays and substrate profiling and was compared to MdpS. Further Mdp characterisation included structural modelling, and functional assays analysing the gene expression during biofilm growth on salivary MUC5B, enzyme-induced biofilm dispersal, and mucus degradation analysed through nanoLC-MS/MS, sedimentation profiling, and microrheology.Results MdpS2 shared conformational homology with MdpS despite low sequence identity and showed greater tolerance to pH and sodium chloride. Both genes were significantly upregulated during late stationary biofilm phase. MdpS and MdpS2 hydrolysed MUC5B extensively, with overlapping but distinct hydrolysis patterns. MdpS2 promoted biofilm dispersal and caused a pronounced reduction in MUC5B size and compactness. Microrheology showed selective modulation of MUC5B-rich mucus by MdpS2, while MdpS affected both MUC5B and MUC5AC networks.Conclusions MdpS and MdpS2 exhibit complementary biochemical and functional profiles, supporting their roles in mucin degradation and biofilm remodelling. These findings advance our understanding of how early colonizing streptococci may interact with mucosal surfaces, influence biofilm dynamics and oral ecology, and suggest potential applications in targeting mucus-related disorders.

Objective: To investigate whether low daily doses of fluoridated milk, as an adjunct to oral hygiene routines with fluoridated toothpaste, can prevent caries development in enamel and dentine in adolescents. Material and methods: Adolescents (mean age 13 years) at three dental clinics in Sweden were enrolled to a randomised clinical trial (RCT) including baseline and 2-year follow-up examinations. The intervention group consumed milk supplemented with fluoride (0.75–1.0 mg) daily, while the control group consumed milk with water. Caries lesion development was assessed visually in line with the international caries detection and assessment system (ICDAS), except for proximal surfaces of premolars and molars which were assessed using a radiological classification system. Outcome measures were decayed, missing and filled surfaces (DMFS) increment, caries lesion arrest and progression. Results: Eighty seven participants in the intervention group and 72 in the control group completed the study. The intervention reduced the incidence of DMFS increment for enamel lesions, but not for dentine lesions. The degree of caries lesion arrest in outer enamel was higher in the intervention group compared to the control group, whereas caries lesion progression was lower in the intervention group. Conclusion: Fluoride exerts an effect on caries lesions in the outer enamel, and fluoridated milk thus can be beneficial to adolescents with such lesions. Trial registration: Clinicaltrials.gov registration number NCT06684405.

Introduction: The identification of salivary molecules that can be associated to dental caries could provide insights about caries risk and offer valuable information to develop caries prediction models. However, the search for a universal caries biomarker has proven elusive due to the multifactorial nature of this oral disease. We have therefore performed a systematic effort to identify caries-associated metabolites and proteins in saliva samples from adolescents that had a caries experience and those that were caries-free. Methods: Quantification of approximately 100 molecules was performed by the use of a wide range of techniques, ranging from nuclear magnetic resonance metabolomics to ELISA, Luminex or colorimetric assays, as well as clinical features like plaque accumulation and gingival index. In addition, simplified dietary and oral hygiene habits questionnaires were also obtained. Results: The caries-free group had significantly lower consumption of sweetened beverages and higher tooth brushing frequency. Surprisingly, very few compounds were found to individually provide discriminatory power between caries-experienced and caries-free individuals. The data analysis revealed several potential reasons that could underly this lack of association value with caries, including differences in metabolite concentrations throughout the day, a lack of correlation between metabolite concentrations in plaque and saliva, or sex-related differences, among others. However, when multiple compounds were combined by multivariate analysis and random forest modeling, a combination of 3-5 compounds were found to provide good prediction models for morning (with an AUC accuracy of 0.87) and especially afternoon samples (AUC = 0.93). Conclusion: While few salivary biomarkers could differentiate between caries-free and caries-experienced adolescents, a combination of markers proved effective, particularly in afternoon samples. To predict caries risk, these biomarkers should be validated in larger cohorts and longitudinal settings, considering factors such as gender differences, and variations in oral hygiene and diet.

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.

MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

ABSTRACT Bacterial metabolism in oral biofilms is comprised of complex networks of nutritional chains and biochemical regulations. These processes involve both intraspecies and interspecies networks as well as interactions with components from host saliva, gingival crevicular fluid, and dietary intake. In a previous paper, a large salivary glycoprotein, mucin MUC5B, was suggested to promote a dental health-related phenotype in the oral type strain of Streptococcus gordonii DL1, by regulating bacterial adhesion and protein expression. In this study, nuclear magnetic resonance-based metabolomics was used to examine the effects on the metabolic output of monospecies compared to dual species early biofilms of two clinical strains of oral commensal bacteria, S. gordonii and Actinomyces naeslundii, in the presence of MUC5B. The presence of S. gordonii increased colonization of A. naeslundii on salivary MUC5B, and both commensals were able to utilize MUC5B as a sole nutrient source during early biofilm formation. The metabolomes suggested that the bacteria were able to release mucin carbohydrates from oligosaccharide side chains as well as amino acids from the protein core. Synergistic effects were also seen in the dual species biofilm metabolome compared to the monospecies, indicating that A. naeslundii and S. gordonii cooperated in the degradation of salivary MUC5B. A better understanding of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is meaningful for understanding oral biofilm physiology and may contribute to the development of future prevention strategies for biofilm-induced oral disease. IMPORTANCE The study of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is of interest for understanding oral microbial adaptation to environmental cues and biofilm maturation. Findings in oral commensals can prove useful from the perspectives of both oral and systemic health of the host, as well as the understanding of general microbial biofilm physiology. The knowledge may provide a basis for the development of prognostic biomarkers, or development of new treatment strategies, related to oral health and disease and possibly also to other biofilm-induced conditions. The study is also an important step toward developing the methodology for similar studies in other species and/or growth conditions.

The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teeth and mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicle of proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which express surface adhesins that bind to specific salivary components, attach to the oral tissues. Further development then proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms, which varies between different sites throughout the oral cavity, is determined by a combination of environmental factors such as the properties of the underlying surface, nutrient availability and oxygen levels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the face of sustained environmental perturbation, however, biofilm homeostasis can break down giving rise to dysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processes involved in biofilm development in oral health and disease, and the requirements for experimental system, microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In this chapter we describe some current and well-established methods used in our laboratory for studying oral bacteria in biofilm models which can be adapted to suit the needs of individual users.

All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

Hypothesis: Among other functions, mucins hydrate and protect biological interfaces from mechanical challenges. Mucins also attract interest as biocompatible coatings with excellent lubrication performance. Therefore, it is of high interest to understand the structural response of mucin films to mechanical challenges. We hypothesized that this could be done with Neutron Reflectometry using a novel sample environment where mechanical confinement is achieved by inflating a membrane against the films. Experiments: Oral MUC5B mucin films were investigated by Force Microscopy/Spectroscopy and Neutron Reflectometry both at solid-liquid interfaces and under mechanical confinement. Findings: NR indicated that MUC5B films were almost completely compressed and dehydrated when confined at 1 bar. This was supported by Force Microscopy/Spectroscopy investigations. Force Spectroscopy also indicated that MUC5B films could withstand mechanical confinement by means of steric interactions for pressures lower than ti 0.5 bar i.e., mucins could protect interfaces from mechanical challenges of this magnitude while keeping them hydrated. To investigate mucin films under these pressures by means of the employed sample environment for NR, further technological developments are needed. The most crit-ical would be identifying or developing more flexible membranes that would still meet certain require-ments like chemical homogeneity and very low roughness. (c) 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Background Salivary mucin MUC5B seems to promote biodiversity in dental biofilms, and thereby oral health, for example, by inducing synergistic ‘mucolytic’ activities in a variety of microbial species that need to cooperate for the release of nutrients from the complex glycoprotein. Knowledge of how early colonizers interact with host salivary proteins is integral to better understand the maturation of putatively harmful oral biofilms and could provide key insights into biofilm physiology. Methods The early oral colonizer Streptococcus gordonii DL1 was grown planktonically and in biofilm flow cell systems with uncoated, MUC5B or low-density salivary protein (LDP) coated surfaces. Bacterial cell proteins were extracted and analyzed using a quantitative mass spectrometry-based workflow, and differentially expressed proteins were identified. Results and conclusions Overall, the proteomic profiles of S. gordonii DL1 were similar across conditions. Six novel biofilm cell proteins and three planktonic proteins absent in all biofilm cultures were identified. These differences may provide insights into mechanisms for adaptation to biofilm growth in this species. Salivary MUC5B also elicited specific responses in the biofilm cell proteome. These regulations may represent mechanisms by which this mucin could promote colonization of the commensal S. gordonii in oral biofilms.

BACKGROUND:To respond and adapt to environmental challenges, prokaryotes regulate cellular processes rapidly and reversibly through protein phosphorylation and dephosphorylation. This study investigates the intracellular proteome and Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii. Intracellular proteins from planktonic cells of S. gordonii DL1 were extracted and subjected to 2D-gel electrophoresis. Proteins in general were visualized using Coomassie Brilliant Blue and T-Rex staining. Phosphorylated proteins were visualized with Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were identified by LC-MS/MS and sequence analysis.RESULTS:In total, sixty-one intracellular proteins were identified in S. gordonii DL1, many of which occurred at multiple isoelectric points. Nineteen of these proteins were present as one or more Ser/Thr/Tyr phosphorylated form. The identified phosphoproteins turned out to be involved in a variety of cellular processes.CONCLUSION:Nineteen phosphoproteins involved in various cellular functions were identified in S. gordonii. This is the first time the global intracellular Ser/Thr/Tyr phosphorylation profile has been analysed in an oral streptococcus. Comparison with phosphoproteomes of other species from previous studies showed many similarities. Proteins that are consistently found in a phosphorylated state across several species and growth conditions may represent a core phosphoproteome profile shared by many bacteria.

HYPOTHESIS:Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure. EXPERIMENTS:Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry. FINDINGS:Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.

Objectives: Approximately 25% of the adolescents in the Scandinavian population are treated with a fixed orthodontic appliance (FOA). Adverse effects such as enamel decalcification (white spot lesions - WSL), seem to affect over 30% of patients. WSL have only a limited ability to improve, thus seriously jeopardising the treatment outcome. The aim of present study was to explore the biofilm phenotype by investigating plaque collected: 1) adjacent to brackets, and 2) in gingival margin of maxillary incisors in adolescents with FOA. Incidence of WSL after treatment was also assessed. Design: In eight adolescent patients treated with FOA, supra-gingival plaque formed on: 1) brackets, and 2) along the gingival margin of the maxillary incisors, was collected after 6-8 months of treatment. The patients were documented before and after treatment by intraoral photos. Plaque samples were tested for glycosidase(fluorogenic substrates) and protease (FITC-labelled casein substrate) activities. The plaque samples were visualised by Live/Dead BacLight stain, following which cells were investigated by confocal scanning laser microscopy. Results: In the collected plaque samples, all enzymes tested displayed small variations in activity between the individuals, except glucosidases, which varied significantly. Four patients developed WSL. The patients displayed higher glucosidase activity in plaque of brackets compared to patients without WSL. In seven patients, plaque at the gingival margin displayed higher protease activity than plaque of brackets. Conclusions: The current study shows two distinct environmentally induced biofilm phenotypes: 1) brackets with higher glucosidase activity, and 2) gingival margin with higher protease activity. Glucosidase activity might thus be used as a putative biomarker for risk of WSL.

Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

BACKGROUND AND OBJECTIVE:High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types.MATERIAL AND METHODS:Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation. Cell number was assessed by counting cells in a Bürker chamber. Intracellular Ca(2+) was monitored by recording Fluo 4-AM fluorescence using a laser scanning confocal microscope. Cellular expression of p33 was determined by western blotting.RESULTS:LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50 ) of 4 μm and a rapid and sustained rise in the intracellular Ca(2+) concentration of osteoblasts, suggesting that LL-37 forms pores in the cell membrane. p33 (10 μm) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca(2+) concentration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonstimulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts.CONCLUSIONS:The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteoblasts from LL-37-induced cell damage in patients suffering from chronic periodontitis associated with high levels of LL-37 locally.

Background Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism. Methods Biofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS. Results Lactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47±0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p<0.01) than that in biofilms formed in nutrient broth (0.4±0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria. Comparison of the surface proteomes of biofilm cells of L. fermentum in a MUC5B environment with those in nutrient broth using two-dimensional electrophoresis and mass spectroscopy, showed that the enhanced proteolytic activity was associated with increased expression of a glycoprotease; O- sialoglycoprotein endopeptidase, as well as chaperone proteins such as DnaK and trigger factor. Conclusions Adhesion to mucin-coated surfaces leads to a shift towards a more protease-active phenotype within L. fermentum biofilms and proteases produced within the biofilms can degrade MUC5B mucins. The enhanced proteolytic activity was associated with an increase in O- sialoglycoprotein endopeptidase on the cell surface. We propose that the upregulation of chaperone proteins in the mucin environment may contribute to the protease-active phenotype through activation of the glycopeptidase. This would represent one way for commensal lactobacilli e.g. L. fermentum to exploit complex substrates in their local environment in order to survive on mucosal surfaces.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

ABSTRACT Enterococcus faecalis strain OG1RF and its (p)ppGpp-deficient Δ relA , Δ relQ , and Δ relA Δ relQ mutants were grown in biofilms and evaluated for growth profiles, biofilm morphology, cell viability, and proteolytic activity. E. faecalis lacking (p)ppGpp had a diminished capacity to sustain biofilm formation over an extended period of time and expressed abundant proteolytic activity.

BACKGROUND AND OBJECTIVE:Whole saliva is a complex mixture of fluids essential for the well-being of the oral hard and soft tissues. Saliva contains numerous antimicrobial proteins that help protect the oral ecosystem from infectious agents. Chronic periodontitis is an infectious chronic inflammatory condition that affects the tooth-supporting structures and leads to their destruction. The aim of the present study was to investigate differences in concentrations of salivary lactoferrin in subjects with and without periodontal disease and correlate these values with clinical variables associated with periodontal disease.MATERIAL AND METHODS:Stimulated whole saliva was collected from 17 subjects with chronic periodontitis and 17 periodontally healthy control subjects. Data relating to bleeding on probing, probing pocket depth and horizontal bone loss were registered. Concentrations of lactoferrin, lysozyme and IgA in stimulated whole saliva were quantified using ELISA.RESULTS:Subjects with chronic periodontits showed higher concentrations of lactoferrin in stimulated whole saliva compared with periodontally healthy control subjects (p < 0.05). Salivary concentrations of lactoferrin were positively correlated with bleeding on probing (p < 0.001) and the number of sites with probing pocket depth ≥ 6 mm (p < 0.001).CONCLUSION:Lactoferrin is raised in stimulated whole saliva in subjects with chronic periodontitis and is correlated with probing pocket depth ≥ 6 mm.