Molecular Oral Microbiology最新文献

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.

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.

Introduction: Filifactor alocis is a newly appreciated member of the periodontal community with a strong periodontal disease correlation. Little is known about the survival mechanisms by which F. alocis copes with oxidative stress and establishes the infection within the local inflammatory microenvironment of the periodontal pocket. The aim of this study is to investigate if F. alocis putative peroxiredoxin/AhpC protein FA768 may constitute an alkyl hydroperoxide reductase system utilizing putative thioredoxin reductase protein FA608, and putative thioredoxin/glutaredoxin homolog FA1411/FA455.

Methods: FA768, FA608, FA1411 and FA455 proteins from F. alocis were expressed and purified from Escherichia coli. Insulin and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reduction assays were performed to determine if purified FA1411 and FA455 proteins could be a substrate for FA608. The peroxidase activity of FA768 was examined by measuring its ability to reduce hydrogen peroxide (H₂O₂) with FA608 and FA1411/FA455 provided as the reducing systems. Further, the hydroperoxide substrate specificity of FA768 was analyzed by monitoring the NADPH oxidation in the presence of different peroxides, including H₂O₂, cumyl hydroperoxide (CHP), and tert-butyl hydroperoxide (t-BHP).

Results: In this study, we have demonstrated the existence of a functioning thioredoxin-dependent alkyl hydroperoxide system in F. alocis. This system is comprised of a thioredoxin reductase (FA608), a thioredoxin/glutaredoxin homolog (FA1411/FA455), and a typical 2-cysteine peroxiredoxin/AhpC (FA768). FA608, together with FA1411/FA455, can function as a thioredoxin reductase system to reduce insulin, DTNB, and FA768. FA455 is a glutaredoxin-like protein with thioredoxin functions in F. alocis. Both the FA768/FA608/FA1411 and FA768/FA608/FA455 reductase systems were NADPH-dependent and exhibited specificity for broad hydroperoxide substrates H₂O₂, CHP, and t-BHP.

Conclusions: This is the first study of a thioredoxin dependent alkyl hydroperoxide system from a periodontal pathogen. This system is proposed to protect F. alocis against oxidative stress due to the likely absence of a catalase or an additional peroxiredoxin homolog.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences capable of editing a host genome sequence. CRISPR and its specific CRISPR-associated (Cas) protein complexes have been adapted for various applications. These include activating or inhibiting specific genetic sequences or acting as molecular scissors to cut and modify the host DNA precisely. CRISPR-Cas systems are also naturally present in many oral bacteria, where they aid in nutrition, biofilm formation, inter- and intraspecies communication (quorum sensing), horizontal gene transfer, virulence, inflammation modulation, coinfection, and immune response evasion. It even functions as an adaptive immune system, defending microbes against invading viruses and foreign genetic elements from other bacteria by targeting and degrading their DNA. Recently, CRISPR-Cas systems have been tested as molecular editing tools to manipulate specific genes linked with periodontal disease (such as periodontitis) and as novel methods of delivering antimicrobial agents to overcome antimicrobial resistance. With the rapidly increasing role of CRISPR in treating inflammatory diseases, its application in periodontal disease is also becoming popular. Therefore, this review aims to discuss the different types of CRISPR-Cas in oral microbes and their role in periodontal disease pathogenesis and precision periodontal therapy.

Objective: To examine the characteristics of the salivary microbiome in Type 2 diabetes mellitus (T2DM) patients with or without periodontitis.

Background: Periodontitis has been identified as clear sequelae of T2DM. This chronic oral disease also impacts the management of the clinical features of diabetes. The oral microbiome characteristics in T2DM with and without periodontitis, as well as the response of this oral microbiome to nonsurgical therapy have not been well described. Knowledge of key oral biological features could help address the observed poorer clinical presentation of T2DM patients.

Methods: The oral microbiome in saliva of adult cohorts periodontally healthy/non-diabetic (non-periodontitis; NP; n = 31), T2DM without periodontitis (DWoP; n = 32), and T2DM with periodontitis (DWP; n = 29) were characterized by microbial molecular analysis using V3-V4 sequencing and Luminex or ELISA techniques for salivary host analytes.

Results: Phyla distribution showed DWP with significantly lower levels of Firmicutes and Actinobacteria and higher levels of Fusobacteria and Spirochetes compared to the healthier groups. Approximately 10% of the detected microbial species showed significant differences in frequency and level of colonization among the DWP, DWoP, and NP samples. A subset of bacteria were significantly correlated with clinical disease features, as well as a specific repertoire of salivary analytes, in particular matrix metalloproteinase (MMP)8/MMP9, interleukin-1ß, B-cell activating factor, and resistin differed between the groups and were related to specific taxa. Principal component analysis that identified a majority of the DWP subjects microbiome was unique based upon an array of 27 taxa out of up to 255 detected in the saliva samples.

Conclusion: T2DM patients with periodontitis show unique oral microbiome and salivary analyte composition compared to diabetics or non-diabetic persons without periodontitis. Specific members of the oral microbiome relate directly to the clinical disease features and/or salivary biomolecules in T2DM individuals.

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.

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.

Background: The PG1037 gene is part of the uvrA-PG1037-pcrA operon in Porphyromonas gingivalis. It encodes for a protein of unknown function upregulated under hydrogen peroxide (H₂O₂)-induced oxidative stress. Bioinformatic analysis shows that PG1037 has a zinc-finger motif, two peroxidase motifs, and one cytidylate kinase domain. The aim of this study is to characterize further the role of the PG1037 recombinant protein in the unique 8-oxoG repair system in P. gingivalis.

Materials and methods: PG1037 recombinant proteins with deletions in the zinc-finger or peroxidase motifs were created. Electrophoretic mobility shift assays were used to evaluate the ability of the recombinant proteins to bind 8-oxoG-containing oligonucleotides. Zinc binding, peroxidase, and Fenton reaction assays were used to assess the functional roles of the rPG1037 protein. A bacterial adenylate cyclase two-bride assay was used to identify the partner protein of PG1037 in the repair of 8-oxoG.

Results: The recombinant PG1037 (rPG1037) protein carrying an N-terminal His-tag demonstrated an ability to recognize and bind 8-oxoG-containing oligonucleotide. In contrast to the wild-type rPG1037 protein, the zinc-finger motif deletion resulted in the loss of zinc and 8-oxoG binding activities. A deletion of the peroxidase motif-1 showed a decrease in peroxidase activity. Using a bacterial adenylate cyclase two-hybrid system, there was no observed protein-protein interaction of PG1037 with UvrA (PG1036), PcrA (PG1038), or mismatch repair system proteins.

Conclusions: Taken together, the results show that PG1037 is an important member of a novel mechanism that recognizes and repairs oxidative stress-induced DNA damage in P. gingivalis.

Fusobacterium nucleatum, a gram-negative anaerobic bacterium abundantly found in the human oral cavity, is widely recognized as a key pathobiont responsible for the initiation and progression of periodontal diseases due to its remarkable aggregative capabilities. Numerous clinical studies have linked F. nucleatum with unfavorable prognostic outcomes in various malignancies. In further research, scholars have partially elucidated the mechanisms underlying F. nucleatum's impact on various types of cancer, thus gaining a certain comprehension of the role played by F. nucleatum in cancer. In this comprehensive review, we present an in-depth synthesis of the interplay between F. nucleatum and different cancers, focusing on aspects such as tumor initiation, metastasis, chemoresistance, and modulation of the tumor immune microenvironment and immunotherapy. The implications for cancer diagnosis and treatment are also summarized. The objective of this review is to enhance our comprehension of the intricate relationship between F. nucleatum and oncogenic pathogenesis, while emphasizing potential therapeutic strategies.