Molecular Oral Microbiology最新文献

Hypothiocyanite (OSCN^-/HOSCN) is an antimicrobial molecule found at high concentrations in saliva. HOSCN is thought to differentially affect oral streptococci, since noncariogenic streptococci (e.g. Streptococcus sanguinis) possess HOSCN reductase activity that cariogenic streptococci (e.g. Streptococcus mutans) lack. However, the enzyme responsible for this activity and the effects of HOSCN and HOSCN reductase activity on biofilm formation by oral streptococci have not been previously established. In this work, we developed an artificial saliva medium for growth of oral streptococci with minimal redox-active components, called Defined Recipe Optimized Oral Liquid (DROOL), and used it to characterize the HOSCN responses of S. sanguinis and S. mutans. We identified a homolog of the Streptococcus pneumoniae Har protein in S. sanguinis as HOSCN reductase. S. mutans wild-type and S. sanguinis ∆har mutants were more sensitive to inhibition by physiological concentrations of HOSCN in DROOL than wild-type S. sanguinis when grown planktonically. S. mutans biofilm formation and glucan production were strongly decreased by HOSCN treatment, suggesting HOSCN inhibits S. mutans exopolysaccharide production. Collectively, our data demonstrate the specific ability of HOSCN to inhibit functions of cariogenic but not noncariogenic oral streptococci and show that Har is responsible for mediating this difference.

Tolerance refers to a hyporesponsiveness toward repeated stimulations with bacteria and their virulence factors, which might exist in the development of periodontitis. To identify the roles of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in periodontitis, an original tolerized mice model was established by high-dose of oral P. gingivalis inoculation following a primary infection. The alveolar bone loss of maxillae was detected by Micro-CT. The infiltration of neutrophils and macrophages, and macrophage polarization were detected by IHC and flow cytometry, respectively. Residual P. gingivalis in subgingival plaque with and without macrophage/neutrophil depletion was measured by real-time PCR. Moreover, a real-time PCR chip and bioinformatic analysis were then employed to explore the cytokine expression profiles in gingivae. The abundance of TNF-α, Toll-like receptor 2 (TLR2), and TLR4 were further verified by western blot. In comparison with the non-tolerance group, TNF-α protein levels, alveolar bone loss, and the infiltration of neutrophils and macrophages in the tolerance group were significantly suppressed (p < 0.05), while the quantities of residual P. gingivalis in subgingival plaque were increased (p < 0.05). Moreover, the depletion of macrophages by liposomal clodronate weakened the inhibitory effect of tolerance, as evidenced by the lack of differences in the quantities of residual bacteria between the tolerance and non-tolerance groups (p > 0.05). Macrophages in gingivae of tolerized mice were more likely to polarize into M2 type. In addition, the expressions of cytokines related to neutrophil and macrophage infiltration and recruitment and the protein levels of TLR2 and TLR4 were decreased in tolerized mice (p < 0.05). Tolerance induced by repeated P. gingivalis stimulations suppressed inflammatory responses in periodontal tissues, and the established periodontal tolerance model provided a reliable tool for the further study on periodontal tolerance in vivo.

Epithelial-mesenchymal transition (EMT) is a fundamental biological process where epithelial cells lose their polarity and adhesion properties, acquiring mesenchymal characteristics such as enhanced migratory ability and invasiveness. Cells undergoing EMT exhibit enhanced motility, aggressiveness, and stemness, contributing to a pro-tumor environment that facilitates malignant metastasis in cancer. Numerous studies have suggested that oral microbes facilitate carcinogenesis through EMT. Oral microbes can directly initiate EMT by adhering to mucosal layers and provoking the disintegration of intercellular adhesion among epithelial cells, thereby modifying cell polarity and downstream signaling pathways. Indirectly, the microbial metabolites and associated compounds can affect the dynamics of EMT. This review summarizes the mechanisms by which oral microbes regulate EMT and thus contribute significantly to cancer. Elucidating the mechanisms underlying the increased plasticity of cancer cells induced by the oral microbiota will facilitate the development of novel targeted therapeutic strategies.

With the growing threat of antimicrobial resistance (AMR), antivirulence strategies present a promising alternative to traditional antibiotics, particularly in dentistry. Dental caries, a chronic biofilm-associated disease primarily driven by the AMR pathogen Streptococcus mutans, results in enamel demineralization and significant oral health challenges. This study explores the anticariogenic mechanism of marine-derived cyclo(l-leucyl-l-prolyl) (CLP), a biomolecule known to inhibit key virulence factors of S. mutans UA159. LC-MS/MS proteomic analysis revealed 30 and 71 significantly regulated proteins following 12 and 24 h of CLP treatment, respectively. Protein-protein interaction and gene ontology analyses demonstrated that CLP downregulates critical virulence proteins related to d-alanylation of lipoteichoic acid (LTA), glucan synthesis, acid production and acid tolerance, while upregulating proteins associated with translation, DNA repair and protein metabolism. KEGG pathway analysis highlighted the involvement of downregulated proteins in key metabolic pathways, including d-alanine metabolism, starch and sucrose metabolism, glycolysis and branched-chain amino acid metabolism. Given the pivotal role of d-alanine metabolism in modulating interconnected virulence pathways, a comparative analysis of in vitro virulence assays-including cell adherence, biofilm formation, acid production and cell surface charge-alongside proteomic data signify that CLP specifically targets the d-alanylation of LTA. This hypothesis was further validated by LTA and d-alanine quantification assays, which confirmed a significant reduction in d-alanine content within LTA after CLP treatment, leading to a marked attenuation of S. mutans cariogenic virulence. Additionally, qPCR and molecular docking analyses corroborated that CLP disrupts S. mutans virulence by interfering with the d-alanylation of LTA. These findings highlight CLP's potential as a novel therapeutic agent for combating dental cariogenesis by targeting S. mutans virulence, offering a promising avenue for the development of advanced anticariogenic therapies.

Multifunctional gingipains are trypsin-like enzymes secreted extracellularly by Porphyromonas gingivalis, which require delicate transit and processing to be activated in different mature forms. This review manages to reconstruct each processing step including the specific cleavage sites and relative proteins or helpers. Errors in any steps can lead to the accumulation of immature gingipains and weaken the virulence of P. gingivalis. Of special note, we emphasize the contribution of new studies to the refinement of the gingipain maturation process and factors that influence their pathogenicity. For example, it is proposed that glutamine cyclase, which is responsible for cyclizing exposed glutamine to pyroglutamic acid after the N-terminal signal peptide is removed, may be able to serve as a potential target for periodontitis treatment, as normal cyclization is key to maintaining the stability of gingipains. Further structural and functional unraveling of the type IX secretion system components, such as the identification of the structure of the PorV-associated shuttle complex, the determination of PorZ's role as the A-LPS deliverer, and the confirmation of the specific mechanism by which PorU promotes CTD removal and catalyzes the transpeptide reaction, has also contributed to a better understanding of gingipain processing. Meanwhile, as the successful activation of gingipains serves to fulfill their functions, this work also concentrates on gingipain pathogenicity, with a particular focus on how gingipains can induce or stimulate the development of systemic diseases, such as causing cardiovascular disorder through vascular damage or exacerbating inflammation in the brain in Alzheimer's disease after crossing the blood-brain barrier.

Periodontitis (periodontal disease [PD]) is a complex inflammatory disease caused by a polymicrobial infection that facilitates the destruction of the connective tissue and bone that support the teeth. PD is highly correlated with cardiovascular disease, low birth weight, preterm osteoporosis, Alzheimer's disease, and rheumatoid arthritis. Porphyromonas gingivalis, a main causative agent of PD, is a non-motile, asaccharolytic, Gram-negative bacterium identified in subgingival, supragingival, and tongue sites in patients. P. gingivalis produces an arsenal of virulence factors, which include fimbriae, lipopolysaccharide (LPS), gingipains and other proteases, P. gingivalis peptidyl arginine deiminase (PPAD), and others. Recently, a number of reports highlighted novel aspects of P. gingivalis virulence. LPS signaling via Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) was elucidated; outer membrane vesicles (OMVs) were implicated as the shuttle for inflammatory induction and neurotoxicity, and gingipains were found to disrupt the integrity of blood-brain barrier (BBB). Further, Tpr protease substrate specificity was described in detail, a novel variant of PPAD was identified and correlated with the aggressive disease, and the role of C-terminal domain as the substrate for the Type IX secretion system (T9SS) transport has been unveiled, together with the identification of the first T9SS inhibitors. The impact of the COVID-19 pandemic prompted the novel research, expanding our understanding of the P. gingivalis correlation with viral infections. These recent findings implicate the need to update the current knowledge of the P. gingivalis virulence factors and provide a comprehensive review of the current trends in P. gingivalis research.

The oral biofilm has been instrumental in advancing microbial research and enhancing our understanding of oral health and disease. Recent developments in next-generation sequencing have provided detailed insights into the microbial composition of the oral microbiome, enabling species-level analyses of biofilm interactions. Fluorescence in situ hybridization (FISH) has been especially valuable for studying the spatial organization of these microbes, revealing intricate arrangements such as "corncob" structures that highlight close bacterial interactions. As more genetic sequence data become available, the specificity and accuracy of existing FISH probes used in biogeographical studies require reevaluation. This study examines the performance of commonly used species-specific FISH probes, designed to differentiate oral microbes within in situ oral biofilms, when applied in vitro to an expanded set of bacterial strains. Our findings reveal that the specificity of several FISH probes is compromised, with cross-species hybridization being more common than previously assumed. Notably, we demonstrate that biogeographical associations within in situ oral biofilms, particularly involving Streptococcus and Corynebacterium, may need to be reassessed to align with the latest metagenomic data.

Background: Oral lichen planus (OLP) is a common T-cell-mediated chronic inflammatory disease of the oral mucosa. Different T-cell subsets play distinct roles in the pathogenesis of OLP. This study aims to reveal the composition and heterogeneity of T cells in the immune microenvironment of OLP using single-cell RNA sequencing (scRNA-seq), thus providing new insights into the pathogenesis of OLP.

Materials and methods: Oral mucosal tissues were collected from three OLP patients and three healthy individuals for scRNA-seq. Data were processed using R software for dimensionality reduction, clustering, annotation, proportion analysis, gene expression visualization, and pseudotime analysis. A chronic inflammation model was established by injecting Prevotella melaninogenica bacteria solution into the buccal mucosa of mice. RT-qPCR was used to detect the expression levels of OLP-related inflammatory factors (Tnf-α, Il-1b, and Il-6) and the exhaustion marker Pd1. HE and immunofluorescence staining were employed to assess histopathological changes in oral mucosal tissues and the quantity of CD8⁺-exhausted T cells (CD8⁺Tex).

Results: ScRNA-seq results showed a significant increase in T cell numbers in the oral mucosal tissues of OLP patients compared to healthy individuals. The average expression levels of effector molecules (GZMB, PRF1, TNFA, IL2, and IFNG) in CD8⁺ T cells were reduced. The number of CD8⁺Tex significantly increased, and these cells were in the terminal stage of CD8⁺ T-cell differentiation, thereby expressing high levels of terminal exhaustion-related genes (PDCD1, LAG3, and TIGIT). Compared to the control group, the P. melaninogenica chronic inflammation group exhibited epithelial thickening and inflammatory cell infiltration in the lamina propria, with significantly upregulated expression of OLP-related inflammatory factors and Pd1. Immunofluorescence staining revealed increased CD8⁺Tex in the oral mucosa of OLP patients and P. melaninogenica mice model.

Conclusions: During the pathogenesis of OLP, the overall ability of T cells to clear antigens is decreased, leading to an inadequate ability to promptly eliminate pathogens and infected cells, which may cause the chronicity of OLP inflammation.

Porphyromonas gingivalis, the bacterium responsible for periodontitis, produces several pathogenic factors, including methyl mercaptan, which contribute to the disease. Kouboku (Magnoliaceae), a Chinese herbal medicine, has been shown to suppress methyl mercaptan production from P. gingivalis. In this study, we investigated the inhibitory effect of Kouboku on methyl mercaptan production, biofilm formation, P. gingivalis-host cell interactions, and its potential synergistic antibacterial effect with antibiotics. Five standard and five clinically isolated P. gingivalis strains were evaluated. Methyl mercaptan production was measured using OralChroma. The mRNA expression of mgl and fimA, which are involved in methyl mercaptan synthesis and adhesion molecules, was assessed using quantitative PCR. Biofilm formation by P. gingivalis and epithelial cell adhesion were analyzed following treatment with or without Kouboku. Furthermore, the effects of the active ingredients of Kouboku, honokiol, and magnolol, on the minimum inhibitory concentrations (MICs) of antibiotics against P. gingivalis were determined. No significant differences were observed in the suppression of methyl mercaptan production among P. gingivalis strains with different FimA genotypes treated with Kouboku. Moreover, Kouboku inhibited biofilm formation in co-cultures of P. gingivalis and Fusobacterium nucleatum, as well as the adhesion of P. gingivalis to gingival epithelial cells through the downregulation of fimA. Treatment with honokiol and magnolol reduced the MICs of ampicillin, gentamicin, erythromycin, and tetracycline against P. gingivalis. These findings demonstrate that Kouboku affects P. gingivalis by modulating its adhesion to other bacteria and host cells, and enhances the antibacterial activity of certain antibiotics.

Periodontal diseases, particularly periodontitis, are complex inflammatory conditions caused by interactions between oral microbiota and the host immune response. Lipopolysaccharides (LPSs) from Gram-negative bacteria like Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis are key in pathogenesis. This review examines how LPS impacts systemic health through direct invasion, compromised oral barriers, increased vascular permeability, and immune cell transport. LPS triggers inflammation in periodontal tissues, leading to tissue destruction and disease progression. In the bloodstream, LPS contributes to conditions, such as cardiovascular diseases, diabetes, respiratory diseases, and rheumatoid arthritis. Current treatments include mechanical debridement, antibiotics, antimicrobial mouthwashes, and anti-inflammatory therapies. Despite progress, gaps remain in understanding the molecular mechanisms of LPS in systemic diseases. Future research should focus on longitudinal studies, the gut-oral axis, biomarkers for early detection, and the lymphatic system's role in LPS dissemination. Maintaining periodontal health is crucial for overall systemic well-being.