Molecular Oral Microbiology最新文献

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.

Background: Periodontitis is caused by a dysbiosis of oral bacteria resulting in alveolar bone destruction and teeth loss. The role of platelets in pathogenesis of periodontitis is a subject of research. The release of toxins from periodontitis-associated bacteria may influence platelet function and contribute to the modulation of hemostatic or inflammatory responses. Therefore, we explored platelet function upon exposure to defined toxins: leukotoxin A from Aggregatibacter actinomycetemcomitans (LtxA), a synthetic version of the C14-Tri-LAN-Gly peptide from Fusobacterium nucleatum (C14), and lipopolysaccharides from Porphyromonas gingivalis (LPS).

Methods: Light transmission aggregometry was performed after the addition of toxins to platelet-rich plasma in different doses. Flow cytometry was used to identify inhibitory effects of toxins by measuring phosphorylation of the vaso-dilator-stimulated phosphoprotein or to identify activating effects by the detection of CD62P expression. The release of chemokines derived from washed platelets was determined by immunoassays.

Results: Collagen-induced threshold aggregation values were diminished upon incubation with LtxA and C14, accompanied with an increase of vaso-dilator-stimulated phosphoprotein (VASP) phosphorylation, indicating platelet inhibition. In contrast, LPS did not affect aggregation but slightly enhanced CD62P expression under co-stimulation with low-dose thrombin pointing to slight platelet activation. The three toxins did not relevantly influence the secretion of chemokines.

Conclusions: Although weak, the investigated toxins differently influenced human platelet function. LtxA and C14 mediated inhibitory effects, whereas LPS contributed to a slight activation of platelets. Further analysis of specific cellular responses mediated by bacterial toxins may render novel targets and suggestions for the treatment of periodontitis.

Background: Type 2 diabetes mellitus (T2DM) may affect the oral microbial community, exacerbating periodontal inflammation; however, its pathogenic mechanisms remain unclear. As nucleotide-binding oligomerization domain 2 (NOD2) plays a crucial role in the activation during periodontitis (PD), it is hypothesized that changes in the oral microbial community due to diabetes enhance periodontal inflammation through the activation of NOD2.

Methods: We collected subgingival plaque from 180 subjects who were categorized into two groups based on the presence or absence of T2DM. The composition of oral microbiota was detected by 16S rRNA high-throughput sequencing. In animal models of PD with or without T2DM, we assessed alveolar bone resorption by micro-computerized tomography and used immunohistochemistry to detect NOD2 expression in alveolar bone. Primary osteoblasts were cultured in osteogenic induction medium with high or normal glucose and treated with inactivated bacteria. After 24 h of inactivated bacteria intervention, the osteogenic differentiation ability was detected by alkaline phosphatase (ALP) staining, and the expressions of NOD2 and interleukin-12 (IL-6) were detected by western blot.

Results: The relative abundance of Parvimonas and Filifactor in the T2DM group was increased compared to the group without T2DM. In animal models, alveolar bone mass was decreased in PD, particularly in T2DM with PD (DMPD) group, compared to controls. Immunohistochemistry revealed NOD2 in osteoblasts from the alveolar bone in both the PD group and DMPD group, especially in the DMPD group. In vitro, intervention with inactivated Parvimonas significantly reduced ALP secretion of primary osteoblasts in high glucose medium, accompanied by increased expression of NOD2 and IL-6.

Conclusions: The results suggest that T2DM leading to PD may be associated with the activation of NOD2 by Parvimonas.

Porphyromonas gingivalis is a keystone pathogen in periodontitis, and Streptococcus sanguinis is an abundant oral commensal bacterium associated with periodontal health. However, the interaction between P. gingivalis and S. sanguinis remains obscure. Here, we established a strategy for high-throughput measurement of the cell number of P. gingivalis in the coculture with S. sanguinis by detecting the concentration of hydrogen sulfate. The interaction between P. gingivalis and over 2000 S. sanguinis single-gene mutants was characterized using this strategy, and several interaction-associated genes in S. sanguinis were determined by detecting more P. gingivalis cells in the coculture with matched S. sanguinis mutants. Three S. sanguinis interaction-associated genes were predicted to be responsible for cysteine metabolism, and the supplementation of exogenous L-cysteine promoted the cell number of P. gingivalis in the coculture with S. sanguinis. Thus, exogenous L-cysteine and the compromised cysteine metabolism in S. sanguinis enhanced the growth of P. gingivalis in the existence of S. sanguinis. Additionally, the interaction between P. gingivalis and other Streptococcus spp. was examined, and S. pneumoniae was the only streptococci that had no inhibition on the cell number of P. gingivalis. In total, this study established a new strategy for high-throughput screening of the interaction between Streptococcus and P. gingivalis and discovered a set of genes in S. sanguinis that impacted the interaction. The influence of exogenous L-cysteine on the interaction between P. gingivalis and S. sanguinis in the oral cavity needs further investigation.

Background: The oral microbiome-dependent nitrate (NO₃ ^-)-nitrite (NO₂ ^-)-nitric oxide (NO) pathway may help regulate blood pressure. NO₂ ^--producing bacteria in subgingival plaque are reduced in relative abundance in patients with untreated periodontitis compared with periodontally healthy patients. In periodontitis patients, the NO₂ ^--producing bacteria increase several months after periodontal treatment. The early effects of periodontal treatment on NO₂ ^--producing bacteria and the NO₃ ^--NO₂ ^--NO pathway remain unknown. The aim of this study was to determine how periodontal treatment affects the oral NO₂ ^--producing microbiome and salivary NO₃ ^- and NO₂ ^- levels over time.

Methods: The subgingival microbiota of 38 periodontitis patients was analysed before (baseline [BL]) and 1, 7 and 90 days after periodontal treatment. Changes in NO₂ ^--producing bacteria and periodontitis-associated bacteria were determined by 16s rRNA Illumina sequencing. Saliva samples were collected at all-time points to determine NO₃ ^- and NO₂ ^- levels using gas-phase chemiluminescence.

Results: A significant increase was observed in the relative abundance of NO₂ ^--producing species between BL and all subsequent timepoints (all p < 0.001). Periodontitis-associated species decreased at all timepoints, relative to BL (all p < 0.02). NO₂ ^--producing species negatively correlated with periodontitis-associated species at all timepoints, with this relationship strongest 90 days post-treatment (ρ = -0.792, p < 0.001). Despite these findings, no significant changes were found in salivary NO₃ ^- and NO₂ ^- over time (all p > 0.05).

Conclusions: Periodontal treatment induced an immediate increase in the relative abundance of health-associated NO₂ ^--producing bacteria. This increase persisted throughout periodontal healing. Future studies should test the effect of periodontal treatment combined with NO₃ ^- intake on periodontal and cardiovascular health.

Biofilms are subjected to many environmental pressures that can influence community structure and physiology. In the oral cavity, and many other environments, biofilms are exposed to forces generated by fluid flow; however, our understanding of how oral biofilms respond to these forces remains limited. In this study, we developed a linear rocker model of fluid flow to study the impact of shear forces on Streptococcus gordonii and dental plaque-derived multispecies biofilms. We observed that as shear forces increased, S. gordonii biofilm biomass decreased. Reduced biomass was largely independent of overall bacterial growth. Transcriptome analysis of S. gordonii biofilms exposed to moderate levels of shear stress uncovered numerous genes with differential expression under shear. We also evaluated an ex vivo plaque biofilm exposed to fluid shear forces. Like S. gordonii, the plaque biofilm displayed decreased biomass as shear forces increased. Examination of plaque community composition revealed decreased diversity and compositional changes in the plaque biofilm exposed to shear. These studies help to elucidate the impact of fluid shear on oral bacteria and may be extended to other bacterial biofilm systems.

Infection is a known cause of abdominal aortic aneurysm (AAA), and matrix metalloproteases-2 (MMP-2) secreted by vascular smooth muscle cells (SMCs) plays a key role in the structural disruption of the middle layer of the arteries during AAA progression. The periodontal pathogen Porphyromonas gingivalis is highly associated with the progression of periodontitis. GroEL protein of periodontal pathogens is an important virulence factor that can invade the body through either the bloodstream or digestive tract and is associated with numerous systemic diseases. Although P. gingivalis aggravates AAA by increasing the expression of MMP-2 in animal studies, the molecular mechanism through which P. gingivalis regulates the expression of MMP-2 is still unknown and requires further investigation. In this study, we first confirmed through animal experiments that P. gingivalis GroEL promotes MMP-2 secretion from vascular SMCs, thereby aggravating Ang II-induced aortic remodeling and AAA formation. In addition, rat vascular SMCs and A7r5 cells were used to investigate the underlying mechanisms in vitro. The results demonstrated that GroEL can promote the interaction between the K639 site of MMP-2 and SUMO-1, leading to MMP-2 SUMOylation, which inhibits the reoccurrence of non-K639-mediated monoubiquitylation. Hence, the monoubiquitylation-mediated lysosomal degradation of MMP-2 is inhibited, consequently promoting MMP-2 stability and production. SUMOylation may facilitate intra-endoplasmic reticulum (ER) and Golgi trafficking of MMP-2, thereby enhancing its transport capacity. In conclusion, this is the first report demonstrating the presence of a novel posttranslational modification, SUMOylation, in the MMP family, suggesting that P. gingivalis GroEL may exacerbate AAA formation by increasing MMP-2 production through SUMOylation in vascular SMCs. This study also provides a novel perspective on the role of SUMOylation in MMP-2-induced systemic diseases.

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.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMBs) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n = 10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the MicrobiomeAnalyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.