Molecular Oral Microbiology最新文献

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.

Actinomyces naeslundii and Schaalia odontolytica belong to the most predominant nitrite-producing bacteria in the oral microbiome. Nitrite has antibacterial and vasodilatory effects that may contribute to maintaining oral and systemic health. We have previously elucidated the metabolic characteristics of the nitrite-producing activity of oral Veillonella species and the effects of oral environmental factors. However, this is still unknown for Actinomyces and Schaalia species. Furthermore, these bacteria are thought to degrade nitrite. Therefore, this study aimed to comprehensively elucidate the effects of environmental factors (pH, oxygen concentration, glucose, lactate, and the presence of nitrate/nitrite during growth) on nitrate and nitrite metabolism of these bacterial species using the type strains. Nitrite was quantified by Griess reagent, and final metabolites were analyzed by high-performance liquid chromatography (HPLC). The nitrite-producing activity of A. naeslundii and S. odontolytica was affected variously by environmental factors. Especially in A. naeslundii, under anaerobic conditions, the activity increased in a concentration-dependent manner with the addition of glucose or lactate and was higher at lower pH when lactate was added. The nitrite-degrading activity of both bacteria was lower than the nitrite-producing activity and was less affected by environmental factors. Metabolites from glucose by A. naeslundii were different with and without nitrate, suggesting that nitrate altered metabolic pathways. The growth was inhibited under anaerobic conditions but promoted under aerobic conditions. These results indicate that the nitrite-producing capacity of the oral microflora must take into account not only the composition and abundance of bacteria but also the variation in metabolic activity due to various environmental factors.

Streptococcus mutans, a key player in dental caries, faces multiple environmental challenges within the oral cavity, including oxidative stress, nutrient scarcity, and acidic pH. To survive and thrive, S. mutans has evolved intricate mechanisms, including the CSP-ComDE quorum sensing system, which coordinates responses to environmental cues. The CSP-ComDE system enables S. mutans to communicate with neighboring cells via its CSP pheromone. Under stress conditions, the CSP pheromone production increases, triggering a cascade of events. Notably, our research demonstrated that the CSP pheromone activates the expression of a Type II restriction-modification (R-M) system. Type II R-M systems are well-known tools in molecular biology and genetic engineering and consist of two distinct enzymes: a restriction enzyme and a methyltransferase. An increasing number of studies have revealed that bacterial adenine methylation (Dam methylation) has a broader role beyond mere DNA protection. In fact, the marks introduced into the DNA provide signals for a variety of physiological processes. Our results highlight a conserved chromosomal locus in S. mutans encoding the DpnII R-M system. DpnII R-M methylates DNA at 5'-GATC target sites within the S. mutans genome and cleaves unmarked DNA. Furthermore, our findings suggest that Dam methylation significantly impacts foreign DNA acquisition via natural transformation and modulates mutanobactin expression-a secondary metabolite linked to oxidative stress tolerance. Collectively, our findings suggest that Dam methylation bridges epigenetics and bacterial fitness, potentially opening new avenues in bacterial epigenetics. As we explore this intricate biological process, we may uncover novel therapeutic strategies to combat bacterial infections.

Streptococcus mutans, the principal pathogen associated with dental caries, impacts individuals across all age groups and geographic regions. Beyond its role in compromising oral health, a growing body of research has established a link between S. mutans and various systemic diseases, including immunoglobulin A nephropathy (IgAN), nonalcoholic steatohepatitis (NASH), infective endocarditis (IE), ulcerative colitis (UC), cerebral hemorrhage, and tumors. The pathogenic mechanisms associated with S. mutans frequently involve collagen-binding proteins (CBPs) and protein antigens (PA) present on the bacterial surface. These components facilitate intricate interactions with the host immune system, thereby potentially contributing to various pathological processes. Specifically, CBP is implicated in the deposition of IgA and complement component C3, which exhibits characteristics reminiscent of IgAN-like lesions through animal models, recent clinical studies suggest a potential involvement of S. mutans in IgAN. In addition, CBP binds to complement component C1q, effectively inhibiting the classical activation pathway of the complement system. In addition, CBP promotes the induction of host cells to produce interferon-gamma (IFN-γ). Furthermore, CBP leads to direct inhibitory effects on platelets and the activation of matrix metalloproteinase-9 (MMP-9) at sites of vascular injury. Moreover, PA enhances the ability of S. mutans to invade hepatic tissue. Through utilization of its PAc, S. mutans excessively produces kynurenine (KYNA), which promotes the development and progression of oral squamous cell carcinoma (OSCC). This article synthesizes the latest advancements in understanding the mechanisms of intricate interactions between S. mutans and various systemic conditions in humans, expanding our perspective beyond the traditional focus on dental caries.

Periodontitis is the most common oral inflammatory disease, contributing to the onset and progression of Alzheimer's disease. However, a full investigation has not been performed on the expression level of amyloid-β (Aβ) peptides in gingival crevicular fluid (GCF) and its effects on oral pathogens. This study aimed to analyze the expression level of Aβ peptides in GCF of patients with periodontitis and the effects of Aβ peptides against common oral pathogens. GCF samples were collected from patients with periodontitis (n = 15) and periodontally healthy people (n = 10). The antimicrobial effects of Aβ peptides were evaluated on four common oral pathogenic strains using an MTT assay, crystal violet staining, fluorescence microscope, and transmission electron microscope. The protein levels of Aβ40 and Aβ42 were upregulated in the GCF of periodontitis group compared with the healthy group. Both Aβ40 and Aβ42 exhibited antimicrobial effects on Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Lactobacillus acidophilus in both planktonic and biofilm conditions. Further, only Aβ40 showed an antimicrobial effect on the Fusobacterium nucleatum. The results of this study demonstrate that Aβ peptides in GCF may be a relevant indicator of periodontitis status. Besides, the antimicrobial peptides derived from Aβ peptides have great potential in periodontal therapy.