E2F1-deficient NOD/SCID mice are an experimental model for dry mouth

Saliva contains a wide variety of secretory proteins, including α-amylase, lysozyme, peroxidase, immunoglobulins, and mucins. Hyposecretion of saliva and consequent dry mouth will lead to severe dental caries, periodontal disease, and mucosal infections, resulting in degrade of quality of life. Polyposia development is one of sign usually seen in dry mouth patients. However, little is reported in dry mouth-model animal regarding the entire process of polyposia development. We investigated the behavior of polyposia in E2F1-deficient non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice, as a dry mouth-model. E2F1-deficient NOD/SCID mice secreted small amount of saliva under the stimulation with a cholinergic agonist, pilocarpine, compared with control mice. The frequency of water intake by E2F-1-deficient NOD/SCID mice was more than that by control mice. These results suggest that E2F-1-deficient NOD/SCID mice show a behavior similar to polyposia and are very useful experimental model of dry mouth patients

Roles of Salivary Components in Streptococcus mutans Colonization in a New Animal Model Using NOD/SCID.e2f1−/− Mice

Streptococcus mutans plays an important role in biofilm formation on the tooth surface and is the primary causative agent of dental caries. The binding of S. mutans to the salivary pellicle is of considerable etiologic significance and is important in biofilm development. Recently, we produced NOD/SCID.e2f1−/− mice that show hyposalivation, lower salivary antibody, and an extended life span compared to the parent strain: NOD.e2f1−/−. In this study we used NOD/SCID.e2f1−/− 4 or 6 mice to determine the roles of several salivary components in S. mutans colonization in vivo. S. mutans colonization in NOD/SCID.e2f1−/− mice was significantly increased when mice were pre-treated with human saliva or commercial salivary components. Interestingly, pre-treatment with secretory IgA (sIgA) at physiological concentrations promoted significant colonization of S. mutans compared with sIgA at higher concentrations, or with human saliva or other components. Our data suggest the principal effects of specific sIgA on S. mutans occur during S. mutans colonization, where the appropriate concentration of specific sIgA may serve as an anti-microbial agent, agglutinin, or an adherence receptor to surface antigens. Further, specific sIgA supported biofilm formation when the mice were supplied 1% sucrose water and a non-sucrose diet. The data suggests that there are multiple effects exerted by sIgA in S. mutans colonization, with synergistic effects evident under the condition of sIgA and limited nutrients on colonization in NOD/SCID.e2f1−/− mice. This is a new animal model that can be used to assess prevention methods for dental biofilm-dependent diseases such as dental caries

Outer Membrane Vesicles of Porphyromonas gingivalis Elicit a Mucosal Immune Response

We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS) O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs). Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen) and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C)], an agonist of Toll-like receptor 3 (TLR3), was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine

Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases

The Impact of Oral Health on Respiratory Viral Infection

Influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV-2) have caused respiratory diseases worldwide. Coronavirus disease 2019 (COVID-19) is now a global health concern requiring emergent measures. These viruses enter the human body through the oral cavity and infect respiratory cells. Since the oral cavity has a complex microbiota, influence of oral bacteria on respiratory virus infection is considered. Saliva has immune molecules which work as the front line in the biophylactic mechanism and has considerable influence on the incidence and progression of respiratory viral infection. Salivary scavenger molecules, such as gp340 and sialic acid, have been reported to exert anti-influenza virus activity. Salivary secretory immunoglobulin A (SIgA) has potential to acquire immunity against these viruses. Biological features of the oral cavity are thought to affect viral infection in respiratory organs in various ways. In this review, we reviewed the literature addressing the impact of oral conditions on respiratory infectious diseases caused by viruses

Role of SCFAs for Fimbrillin-Dependent Biofilm Formation of Actinomyces oris

Actinomyces oris expresses type 1 and 2 fimbriae on the cell surface. Type 2 fimbriae mediate co-aggregation and biofilm formation and are composed of the shaft fimbrillin FimA and the tip fimbrillin FimB. Short-chain fatty acids (SCFAs) are metabolic products of oral bacteria, but the effects of exogenous SCFAs on FimA-dependent biofilm formation are poorly understood. We performed two types of biofilm formation assays using A. oris MG1 or MG1.ΔfimA to observe the effects of SCFAs on FimA-dependent biofilm formation in 96-well and six-well microtiter plates and a flow cell system. SCFAs did not induce six- and 16-hour biofilm formation of A. oris MG1 and MG1.ΔfimA in saliva-coated 96-well and six-well microtiter plates in which metabolites produced during growth were not excluded. However, 6.25 mM butyric acid and 3.125 mM propionic acid induced FimA-dependent biofilm formation and cell death in a flow cell system in which metabolites produced during growth were excluded. Metabolites produced during growth may lead to disturbing effects of SCFAs on the biofilm formation. The pure effects of SCFAs on biofilm formation were induction of FimA-dependent biofilm formation, but the stress responses from dead cells may regulate its effects. Therefore, SCFA may play a key role in A. oris biofilm formation

Effects of human saliva and salivary components in <i>S. mutans</i> colonization.

<p>Colony numbers of <i>S. mutans</i> in (A) NOD/SCID wild type, (B) NOD/SCID.<i>e2f1</i>^+/−, (C) NOD/SCID.<i>e2f1</i>^−/− female mice, 4 months of age, at 120 min after inoculation. All mice were pre-treated with human saliva or salivary components prior to bacterial inoculation. Data are expressed as the means ± SDs of the results for 6 mice per strain (*<i>P</i><0.05, ** <i>P</i><0.01).</p

Colonization of <i>S. mutans</i> in human-saliva treated mice.

<p>Colony numbers of <i>S. mutans</i> in (A) NOD/SCID wild type, (B) NOD/SCID.<i>e2f1</i>^+/−, (C) NOD/SCID.<i>e2f1</i>^−/− female mice, and 4 months of age pre-treated with and without human saliva prior to bacterial inoculation. Asterisks show significant differences (vs. untreated group, *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001). (D) Time-course analysis of <i>S. mutans</i> colonization for each mouse strain pre-treated with human saliva prior to bacterial inoculation. Data were obtained from three independent experiments with 4 mice from each strain, and values are expressed as the means ± standard daviations (SDs) of the data (*<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, represents significant differences vs. 90 min, <i>P</i><0.05).</p

Molecular Interactions of Surface Protein Peptides of Streptococcus gordonii with Human Salivary Components

Oral streptococci play a large role in dental biofilm formation, and several types interact as early colonizers with the enamel salivary pellicle to form the primary biofilm, as well as to incorporate other bacteria on tooth surfaces. Interactions of surface molecules of individual streptococci with the salivary pellicle on the tooth surface have an influence on the etiological properties of an oral biofilm. To elucidate the molecular interactions of streptococci with salivary components, binding between surface protein (SspB and PAg) peptides of Streptococcus gordonii and Streptococcus sobrinus were investigated by utilizing BIAcore biosensor technology. The analogous peptide [change of T at position 400 to K in SspB(390-402), resulting in the SspB(390-T400K-402) peptide] from S. gordonii showed the greatest response for binding to salivary components and inhibited the binding of Streptococcus sanguis by more than 50% in a competitive inhibition assay in a comparison with other SspB and PAg peptides. This peptide also bound to the high-molecular-weight protein complex of salivary components and the agglutinin (gp340/DMBT1) peptide (scavenger receptor cysteine-rich domain peptide 2 [SRCRP 2]). In addition, the SspB(390-T400K-402) peptide was visualized by two surface positive charges in connection with the positively charged residues, in which lysine was a key residue for binding. Therefore, the region containing lysine may have binding activity in S. gordonii and S. sanguis, and the SRCRP 2 region may function as a receptor for the binding. These findings may provide useful information regarding the molecular mechanism of early biofilm formation by streptococci on tooth surfaces