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.

INTRODUCTION: Salivary 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.

METHODS: To 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 DISCUSSION: MUC5B 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.

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.

BACKGROUND: Lifestyle- and sucrose-dependent polymicrobial ecological shifts are a primary cause of caries in populations with high caries prevalence. In populations with low prevalence, PRH1, PRH2 susceptibility and resistance phenotypes may interact with the Streptococcus mutans adhesin cariogenicity phenotype to affect caries progression, but studies are lacking on how these factors affect the microbial profile of caries.

METHODS: We analysed how the residency and infection profiles of S. mutans adhesin (SpaP A/B/C and Cnm/Cbm) phenotypes and commensal streptococci and lactobacilli influenced caries progression in a prospective case-referent sample of 452 Swedish adolescents with high (P4a), moderate (P6), and low (P1) caries PRH1, PRH2 phenotypes. Isolates of S. mutans from participants were analysed for adhesin expression and glycosylation and in vitro and in situ mechanisms related to caries activity.

FINDINGS: Among adolescents with the resistant (P1) phenotype, infection with S. mutans high-virulence phenotypes was required for caries progression. In contrast, with highly (P4a) or moderately (P6) susceptible phenotypes, caries developed from a broader polymicrobial flora that included moderately cariogenic oral commensal streptococci and lactobacilli and S. mutans phenotypes. High virulence involved unstable residency and fluctuating SpaP ABC, B-1, or Cnm expression/glycosylation phenotypes, whereas low/moderate virulence involved SpaP A phenotypes with stable residency. Adhesin phenotypes did not display changes in individual host residency but were paired within individuals and geographic regions.

INTERPRETATION: These results suggest that receptor PRH1, PRH2 susceptibility and resistance and S. mutans adhesin virulence phenotypes specify different microbial profiles in caries.

FUNDING: Swedish Research Council and funding bodies listed in the acknowledgement section.

INTRODUCTION: Formative assessment with emphasis on feedback has been linked to developmental purposes of assessment, whilst summative assessment is assumed to focus on judgemental and quality assurance purposes. This dichotomy is questioned but designs to blend formative and summative assessments in constructive ways are rare in health care education.

METHODS: We have designed an assessment model blending formative and summative assessments . In the formative assessment at the end of a course, students' responses to real-life scenarios with questions demanding responses at the relational level of understanding were assessed at three levels of understanding (incorrect, descriptive, and relational) modified after the SOLO taxonomy. Students were presented with individual feedback for each response. At the summative assessment of a subsequent course, the students' new responses were assessed underpinning a final judgement of students' performance. The assessment model was justified across three student cohorts.

RESULTS: Both formative and summative assessment events of the model provided information about the levels of understanding, unique to each student. A comparison of results from the assessments demonstrated that most responses developed to a higher level of understanding. With the summative assessment it was possible to make judgements about whether or not individual students passed the pre-set standards.

CONCLUSIONS: We argue that the current assessment model presents real interdependence between formative and summative assessments and can provide information that meets the needs of students as learners, education institutes, and health care organisations. The SOLO taxonomy can be used to emphasise the importance of developing and assessing cognitive complexity.

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.

The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teethand mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicleof proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which expresssurface adhesins that bind to specific salivary components, attach to the oral tissues. Further developmentthen 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 ofenvironmental factors such as the properties of the underlying surface, nutrient availability and oxygenlevels, 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 faceof sustained environmental perturbation, however, biofilm homeostasis can break down giving rise todysbiosis, 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 processesinvolved 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 thischapter we describe some current and well-established methods used in our laboratory for studying oralbacteria in biofilm models which can be adapted to suit the needs of individual users. 

Periodontitis (gum disease) is a common biofilm-mediated oral condition, with around 7% of the adult population suffering from severe disease with risk for tooth loss. Moreover, periodontitis virulence markers have been found in atherosclerotic plaque and brain tissue, suggesting a link to cardiovascular and Alzheimer’s diseases. The lack of accurate, fast, and sensitive clinical methods to identify patients at risk leads, on the one hand, to patients being undiagnosed until the onset of severe disease and, on the other hand, to overtreatment of individuals with mild disease, diverting resources from those patients most in need. The periodontitis-associated bacterium, Porphyromonas gingivalis, secrete gingipains which are highly active proteases recognized as key virulence factors during disease progression. This makes them interesting candidates as predictive biomarkers, but currently, there are no methods in clinical use for monitoring them. Quantifying the levels or proteolytic activity of gingipains in the periodontal pocket surrounding the teeth could enable early-stage disease diagnosis. Here, we report on a monitoring approach based on high-affinity microcontact imprinted polymer-based receptors for the Arg and Lys specific gingipains Rgp and Kgp and their combination with surface plasmon resonance (SPR)-based biosensor technology for quantifying gingipain levels in biofluids and patient samples. Therefore, Rgp and Kgp were immobilized on glass coverslips followed by microcontact imprinting of poly-acrylamide based films anchored to gold sensor chips. The monomers selected were N-isopropyl acrylamide (NIPAM), N-hydroxyethyl acrylamide (HEAA) and N-methacryloyl-4-aminobenzamidine hydrochloride (BAM), with N,N′-methylene bis(acrylamide) (BIS) as the crosslinker. This resulted in imprinted surfaces exhibiting selectivity towards their templates high affinity and selectivity for the templated proteins with dissociation constants (Kd) of 159 and 299 nM for the Rgp- and Kgp-imprinted, surfaces respectively. The former surface displayed even higher affinity (Kd = 71 nM) when tested in dilute cell culture supernatants. Calculated limits of detection for the sensors were 110 and 90 nM corresponding to levels below clinically relevant concentrations.

BACKGROUND: Multivariable prediction models are used in oral health care to identify individuals with an increased likelihood of caries increment. The outcomes of the models should help to manage individualized interventions and to determine the periodicity of service. The objective was to review and critically appraise studies of multivariable prediction models of caries increment.

METHODS: Longitudinal studies that developed or validated prediction models of caries and expressed caries increment as a function of at least three predictors were included. PubMed, Cochrane Library, and Web of Science supplemented with reference lists of included studies were searched. Two reviewers independently extracted data using CHARMS (Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies) and assessed risk of bias and concern regarding applicability using PROBAST (Prediction model Risk Of Bias ASessment Tool). Predictors were analysed and model performance was recalculated as estimated positive (LR +) and negative likelihood ratios (LR -) based on sensitivity and specificity presented in the studies included.

RESULTS: Among the 765 reports identified, 21 studies providing 66 prediction models fulfilled the inclusion criteria. Over 150 candidate predictors were considered, and 31 predictors remained in studies of final developmental models: caries experience, mutans streptococci in saliva, fluoride supplements, and visible dental plaque being the most common predictors. Predictive performances varied, providing LR + and LR - ranges of 0.78-10.3 and 0.0-1.1, respectively. Only four models of coronal caries and one root caries model scored LR + values of at least 5. All studies were assessed as having high risk of bias, generally due to insufficient number of outcomes in relation to candidate predictors and considerable uncertainty regarding predictor thresholds and measurements. Concern regarding applicability was low overall.

CONCLUSIONS: The review calls attention to several methodological deficiencies and the significant heterogeneity observed across the studies ruled out meta-analyses. Flawed or distorted study estimates lead to uncertainty about the prediction, which limits the models' usefulness in clinical decision-making. The modest performance of most models implies that alternative predictors should be considered, such as bacteria with acid tolerant properties.

TRIAL REGISTRATION: PROSPERO CRD#152,467 April 28, 2020.

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.