The oral-derived pathobiont Fusobacterium nucleatum has increasingly been implicated in colorectal cancer (CRC) progression through its ability to modulate inflammatory, immunological, and molecular pathways. F. nucleatum has emerged as a key oral-derived pathobiont frequently associated with tumor aggressiveness and alterations in the colorectal tumor microenvironment. This review critically examines recent mechanistic evidence from in vitro, in vivo, and advanced experimental models to elucidate how F. nucleatum interacts with host pathways involved in colorectal carcinogenesis. Available evidence indicates that this bacterium actively reshapes the tumor microenvironment through coordinated effects on immune signaling, epigenetic regulation, metabolic adaptation, and metastatic competence. Mechanistically, F. nucleatum promotes sustained inflammatory activation, macrophage polarization, epithelial-mesenchymal transition, endothelial remodeling, and resistance to regulated cell death pathways, while also influencing transcriptional and oncogenic programs associated with tumor progression. Emerging evidence further suggests that microbial effects may be modulated by tumor heterogeneity and long-term environmental exposures, reinforcing the complexity of host-microbiome interactions in CRC. Collectively, the findings support the concept that F. nucleatum functions as an active biological driver rather than a passive microbial bystander in CRC progression. These insights highlight the translational relevance of tumor-associated microbiota and support the need for approaches combining mechanistic, microbial, and clinical data to refine precision strategies at the tumor-microbiome interface.
Streptococcus mutans is one of the primary causes of dental caries and is often found in persistent endodontic infections, primarily due to its ability to form acidogenic, aciduric and therapeutically recalcitrant biofilms. The VicRK and LiaSR two-component signal transduction systems (TCS) are particularly important regulators of virulence, envelope integrity and stress response. This paper will provide an updated synthesis of the evidence, at the molecular, genetic and systems levels, regarding the architecture, regulation and integration of VicRK and LiaSR in the control of S. mutans pathogenicity. VicRK helps synthesise essential molecules for biofilm formation and reduces the negative impact of environmental stress on microbes. At the same time, LiaSR responds to antimicrobial peptides, oxidative stress, detergents and root canal irrigants by activating membrane repair and protective stress pathways. Structural, transcriptomic and proteomic studies now indicate extensive regulatory overlap, including overlapping promoter targets, cross-phosphorylation and coordinate regulation of autolysins, cell wall biosynthetic enzymes and extracellular DNA (eDNA) release. These systems all work together as part of the signalling. The ability of S. mutans to survive different pHs, oxidative bursts, lack of nutrients and chemical disinfection in and outside the mouth. According to mutant, knockout and multi-omics studies, these TCSs are critical for biofilm formation, EPS structures, antimicrobial tolerance and interspecies interactions, including synergistic virulence in mixed-species biofilms. As interest in anti-virulence therapeutics grows, drug targets VicRK and LiaSR have emerged. Potential candidates for the selective disruption of TCS signalling that are unlikely to induce resistance include small-molecule inhibitors, marine-derived bioactive compounds and computationally designed ligands. The review also highlights translational gaps and stresses the need for advanced delivery platforms, clinical validation and sustainable production of natural inhibitors. By integrating knowledge of the mechanisms underlying TCS-mediated virulence with available strategies to address it, this review offers a comprehensive overview of this topic.
Nonhuman primates have been shown to develop periodontitis with clinical and demographic features similar to humans. This preclinical disease model is well-positioned to advance the elucidation of novel biologic-based preventive and therapeutic approaches to controlling periodontitis, a chronic infection and immunoinflammatory disease. In this analysis, the oral microbiomes of younger (10-19 human years; n = 30) nonhuman primates are compared for matriline (an indicator of heritability), sex, and age variations. A holistic assessment of the microbiome similarities/differences suggested two primary conclusions in this younger group of orally healthy nonhuman primates. First, at the microbiome level of phyla, orders, and families, the ecology was relatively similar across sexes, matrilines, and age groups. However, at the genus level, matriline, sex, or age differences were observed. Of interest was that the principal differential genus proportions with matriline and age were similar, but somewhat unique with the sex comparison. These genus differences encompassed microorganisms generally considered as human commensals, albeit Fusobacterium, Tannerella, and Treponema genera did show some variation. The second, broader observation was the rather extensive species variation across these nonhuman primates. Nevertheless, the data could define a "core microbial species" pattern that included species across the Actinobiota, Bacteroidota, Desulfobacterota, Firmicutes/Bacillota, Fusobacteriota, Proteobacteriota, and Spirochaetota phyla. The results provide seminal details of the oral microbiome in this disease model and underpin the ability to elucidate specific microbial changes that can occur related to early-life oral environmental stimuli that may presage a greater risk for periodontitis in adulthood.
The dual-species biofilms formed by Streptococcus mutans and Candida albicans exhibit enhanced cariogenic potential due to the production of extracellular polysaccharides (EPSs). The response regulator GcrR in S. mutans negatively regulates EPS synthesis, but its impact on dual-species biofilms remains unclear. The objective of the present study was to investigate the effect of GcrR on the cariogenicity of cross-kingdom biofilms established by S. mutans and C. albicans. The crystal violet (CV) staining was conducted to evaluate the dual-species biofilm formation. The anthrone-sulfuric acid assay was employed to assess EPS formation. The biofilm morphology was visualized through scanning electron microscopy (SEM), and the structure was examined by confocal laser scanning microscopy (CLSM). The expression levels of genes were determined by quantitative real-time PCR (qRT-PCR). The animal assay was conducted to elucidate the anti-cariogenic effect of GcrR. The overexpression of GcrR suppressed dual-species biofilm formation, which was accompanied by a reduction in both water-soluble glucans (WSGs) and water-insoluble glucans (WIGs). The morphological transformation of C. albicans from yeast to hyphal form was inhibited by GcrR. Expression of gtfB/D and gbpB/C genes in S. mutans and Ras1-cAMP/PKA pathway genes in C. albicans was downregulated. Furthermore, we found that GcrR attenuated the virulence of dual-species biofilms. This study suggests that GcrR in S. mutans plays a critical role in modulating the cariogenic potential of bacterial-fungal biofilms.
Oral commensal streptococci play a large role in mediating microbial homeostasis. Our prior work has demonstrated that hydrogen peroxide (H2O2) produced by oral commensal streptococci can react with salivary nitrite (NO2) and generate peroxynitrite (ONOO-), a potent antimicrobial. We have shown that the combinatorial antimicrobial effects of NO2 and H2O2 produced from the commensal Streptococcus parasanguinis inhibit the colonization and pathogenesis of oral pathogens to promote homeostasis. Remarkably, S. parasanguinis is highly resistant to NO2 and ONOO-. However, it remains unclear how S. parasanguinis tolerates nitrosative stress. The goal of this study was to identify mechanisms used by S. parasanguinis to resist NO2-mediated nitrosative stress. Transcriptomics analysis revealed that the DNA uptake gene, comEA was upregulated during growth on 2 mM NaNO2. Further, loss of the ComEA/ComEC DNA uptake locus resulted in increased sensitivity to peroxynitrite and a decrease in biofilm development, intracellular nitrite transport, H2O2 production, and was defective for colonization in a Drosophila melanogaster colonization model. In summary, our data show that the ComEA/EC DNA uptake operon is critical for key aspects of S. parasanguinis physiology and potentially impacts S. parasanguinis's ability to colonize and modulate homeostasis in the oral cavity.
BACKGROUND:Globally prevalent periodontitis, an inflammatory disease, may be associated with glucose metabolism disorders. Pyruvate kinase M2 (PKM2), the key enzyme of glycolysis, is potentially implicated in periodontitis pathogenesis, although its precise role is unclear. Ginsenoside compound K (CK) has strong anti-inflammatory effects. Prior studies indicate that CK down-regulates PKM2 expression, yet it remains unclear whether CK can regulate PKM2 to modulate the inflammatory response. Furthermore, CK's role in periodontitis lacks pharmacological investigation. METHODS:A periodontitis cell model was established by stimulating human gingival epithelial cells (HGECs) with Porphyromonas gingivalis (Pg), and subsequently intervened with CK. Western blotting assessed the expressions of PKM2, phospho-PKM2 (Tyr105), nuclear factor kappa-B P65 (NF-κB P65), and phosphorylated NF-κB p65 (Ser536). Enzyme-linked immunosorbent assay measured interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in culture supernatants. RESULTS:Pg stimulation induced NF-κB p65 (Ser536) activation and the secretion of IL-1β and TNF-α in HGECs and promoted PKM2 (Tyr105) phosphorylation. PKM2 was found to be the upstream regulator of NF-κB p65, and NF-κB p65 activated PKM2 through positive feedback to promote IL-1β and TNF-α production, forming a feedback regulatory loop. CK inhibited Pg-induced inflammation and NF-κB p65 (Ser536) and PKM2 (Tyr105) phosphorylation in HGECs. Furthermore, PKM2 overexpression significantly reversed CK's anti-inflammatory effects. CONCLUSIONS:The PKM2/NF-κB p65 pathway is involved in regulating the expression of inflammatory cytokines in Pg-stimulated HGECs and serves as a crucial inflammatory regulatory pathway in the Pg-stimulated HGECs model. CK exhibits anti-inflammatory activity by inhibiting the PKM2-mediated NF-κB signaling pathway and has potential as PKM2 modulator for treating periodontitis and other inflammatory diseases.
BACKGROUND:Given the pivotal role of macrophages in both the progression and regenerative phases of periodontitis, targeting macrophage-mediated immunity represents a novel therapeutic strategy for periodontitis management. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) has been previously implicated in inflammatory processes. However, how UCHL1 regulates macrophage immune response in periodontitis remains to be elucidated. METHODS:Ligature-induced periodontitis mice and Porphyromonas gingivalis lipopolysaccharide (P.g LPS) were used to investigate the expression of UCHL1 in macrophages during immune responses. Lentivirus and an inhibitor were conducted to determine the role of UCHL1 in the periodontitis-associated macrophage immune response. The mitochondrial function and the endoplasmic reticulum status of macrophages were examined to elucidate the underlying mechanisms. Finally, we constructed hydrogels containing UCHL1 inhibitor and evaluated their efficacy in treating periodontitis. RESULTS:The expression of UCHL1 was upregulated in proinflammatory immune response of periodontitis-associated macrophages. Inhibition of UCHL1 reduced the expression and release of TNF-α, IL-6, and IL-1β. In addition, inhibition of UCHL1 in macrophages improved the osteogenesis and migration ability of MC3T3-E1 cells in the co-culture system. Mechanistically, inhibition of UCHL1 ameliorated LPS-induced mitochondrial damage and endoplasmic reticulum stress in macrophages. LDN@GelMA hydrogel had great in vitro anti-inflammatory and bone-promoting effects. Periodontal injection of LDN@GelMA hydrogel alleviated local inflammation and promoted alveolar bone regeneration. CONCLUSIONS:Our data suggest that UCHL1 may serve as a critical negative regulator for periodontitis treatment, and inhibition of UCHL1 is expected to improve the periodontal inflammatory microenvironment and promote alveolar bone regeneration.
Porphyromonas gingivalis (Pg), a key pathogen in periodontal disease, is suggested to be involved in the progression of Alzheimer's disease (AD); however, the molecular mechanism remains unclear. We previously reported that Pg-released outer membrane vesicles (OMVs) were detected in the brains of mice after intraperitoneal administration. We here investigated the effects of Pg OMVs on astrocytes, the most abundant glial cells in the central nervous system, which are involved in neuroinflammation. We demonstrated that Pg OMVs increased the expression of interleukin-6 (IL-6) mRNA, which is associated with the pathogenesis of AD, in a Toll-like receptor (TLR)2/4-independent manner in human astrocyte SVG p12 cells. Whole-genome sequencing revealed that Pg OMVs contained Pg genomic DNA, which was critical for IL-6 mRNA induction. The tracking of fluorescent-labeled Pg OMV-associated DNA revealed that Pg OMVs were transported into SVG p12 cells. Endocytosis inhibitors attenuated IL-6 mRNA expression induced by Pg OMVs, suggesting that the incorporation of Pg OMVs by endocytosis is important for IL-6 mRNA induction. Furthermore, the incorporated Pg OMV-associated DNA increased IL-6 mRNA expression via the TLR9 pathway. Our study advances understanding of the role of Pg OMVs, which may contribute to the onset and progression of AD in periodontal disease.
Streptococcus mutans is a key cariogenic pathogen of dental caries due to its strong ability to synthesize extracellular glucans and form biofilms. Glucosyltransferases, encoded by gtfB/C/D genes in S. mutans, are responsible for producing biofilm exopolysaccharides (EPS) and are considered to be critical virulence factors. Previous studies have highlighted the roles of various regulatory factors of gtf genes in S. mutans. Here, we investigated the role of the global transcriptional regulator CcpA encoded by ccpA in regulating the EPS synthesis and biofilm formation of S. mutans. A ccpA in-frame deletion strain was observed to develop shiny, round colonies and longer cell length. In addition, the deletion of ccpA resulted in impaired growth, diminished synthesis of EPS, and reduced biofilm formation. Transcriptome analysis revealed that differentially expressed genes in the ccpA deletion strain were significantly enriched in pathways of carbohydrate transport and metabolism, in which the expressions of gtfB and gtfC were downregulated markedly. Electrophoretic mobility shift assays confirmed that CcpA directly binds to the promoter sequences of gtfB and gtfC, with a higher affinity for gtfC. Moreover, the expression level of ccpA in part explained differences in the ability to synthesize sufficient EPS and form stable biofilm in clinically isolated strains. These findings highlight that CcpA plays a crucial role in the EPS production and biofilm formation of S. mutans through directly binding to the promoter regions of gtfB and gtfC. This study provides novel insights into the pathogenic mechanisms of S. mutans and potential strategies for the prevention and treatment of dental caries.
Porphyromonas gingivalis is a key pathogen in periodontitis, with secreted proteases as major virulence factors. We developed a screening method to generate and identify P. gingivalis mutants with elevated protease activity. Mutations were induced using the mutagens 2,6-diaminopurine (2,6-DAP) or ethyl methanesulfonate (EMS), and the mutagenized cells were subsequently plated on casein agar. During colony growth, the medium became opaque due to partial casein precipitation, whereas colonies with higher protease activity produced clear halos through casein degradation. Colonies that formed halos earlier than the wild type were selected for further analysis. Liquid culture assays of the supernatants identified four strains with enhanced protease activity, of which two were 2,6-DAP-derived and two were EMS-derived. Whole-genome sequencing revealed that the two 2,6-DAP-derived strains carried mutations in iron transport-related genes (foeA and tonB, respectively), likely increasing protease levels through iron limitation-induced upregulation of rgpA. The two EMS-derived strains contained multiple mutations, including one in rgpA, a major protease gene. The N-terminal region of RgpA, which contains the protease motif, harbored the G450D mutation in one strain and the C600Y mutation in the other. These results demonstrate that our method efficiently generates P. gingivalis mutants with protease gene alterations that increase enzymatic activity. This approach provides a useful tool for studying protease function and virulence mechanisms in this pathogen, and for identifying genes that affect protease secretion.
The morphological and viscoelastic properties of blood neutrophils and neutrophils isolated from the oral cavity of the same donor were compared using scanning ion conductance microscopy. It was found that cell morphology, including morphometric parameters (height, diameter, and cell volume), did not differ significantly. However, the elasticity of the membrane-cytoskeletal complex was primarily determined by the cell compartment and its functional state (e.g., migration or adhesion) rather than the neutrophils' ecological niche (blood or oral cavity). The ability of blood neutrophils and oral neutrophils to produce reactive oxygen species (ROS) was assessed using luminol-dependent chemiluminescence. Neutrophils isolated from the oral cavity demonstrated a significantly higher ROS-producing activity compared to blood neutrophils from the same donor. Additionally, oral neutrophils exhibited high variability in ROS production levels, even within the same donor on different days of collection. When comparing the stimulation of ROS production in blood and oral neutrophils exposed to S. aureus 2879M, the cells displayed opposing responses: blood neutrophils were stimulated by Staphylococci, whereas oral neutrophils were inhibited. This response persisted when cells were stimulated by Staphylococci preincubated with saliva. The sequential addition of unfiltered saliva to neutrophils and Staphylococci enhanced this trend, while filtered oral fluid attenuated it. For the first time, it was established that saliva without bacteria (filtered through bacterial filters) suppresses the production of ROS by both blood neutrophils and oral neutrophils, most likely indicating an anti-inflammatory effect of saliva.
Bacteria produce membrane vesicles (MVs) in response to environmental stress and genetic changes. Previous studies have shown that MVs can trigger inflammatory responses and may serve as important mediators of host-microbe interactions. Given the dynamic nature of the oral microbiome, bacteria such as Streptococcus mutans are frequently exposed to environmental fluctuations that could alter MV production. The objective of this study was to investigate whether inducing stress conditions would affect MV production and morphology in S. mutans, a prominent oral pathogen. Cultures were subjected to different pH conditions to mimic environmentally relevant stress. MVs were isolated and purified in order to characterize and assess changes in yield, size, and cargo. Our findings show that acidic stress significantly increased MV production while reducing average MV size. We also observed significant differences in MV content when compared to control conditions. These changes may reflect bacterial adaptation strategies and could influence how MVs interact with host immune systems. Overall, this study highlights the potential for environmental stress to reshape MV-mediated communication in the oral microbiome and provides a foundation for exploring how such changes may contribute to inflammation and oral disease.
Socransky's complexes have identified a range of bacteria as key contributors to the onset and progression of periodontal disease. However, advancements in microbiological detection methods have allowed for exploration of the microbiome in periodontal health/disease in greater detail. In recent years, Filifactor alocis has emerged as a potential periodontal pathogen. Therefore, the aim of this review was to investigate whether this bacterium could be included in Socransky's model by summarizing the available evidence. A comprehensive literature search performed using PubMed, ScienceDirect, and Scopus databases was undertaken. The retrieved articles were filtered according to defined eligibility criteria, which yielded 24 studies. Data were extracted from these observational and clinical studies to synthesize findings. Findings regarding the host immune response were derived from in vitro and experimental animal models and narratively summarized. Observational studies and clinical trials showed heterogeneity and a lack of standardized outcomes. However, the general trend indicated a higher prevalence of F. alocis at diseased sites than at healthy sites. In addition, periodontal treatment was found to significantly reduce F. alocis levels and was associated with improvements in clinical periodontal parameters. Experimental models and in vitro studies showed that F. alocis exhibits a range of virulence attributes and pathogenic behavior similar to that of putative pathogenic periodontal bacteria. The evidence is not sufficient to include F. alocis as a new member of Socransky's model. However, this review suggests that this bacterium has the potential to be included in Socransky's complexes in the future after further research which would require to be highly standardized to enhance comparability and generalizability of findings.
Selenomonas sputigena is an understudied oral pathobiont associated with periodontitis and dental caries. We recently demonstrated that S. sputigena binds to gingival epithelial cells (GECs), where afterwards the bacterium causes robust in vitro pro-inflammatory cytokine production and migration of monocytes and neutrophils. Here, we report that extracellular adherent S. sputigena are subsequently internalized by GECs. Fluorescently labeled intracellular S. sputigena were observed in two gingival keratinocyte cell lines and primary cells. Oxygen-killed S. sputigena was found within GECs at a similar rate to living bacteria, suggesting active protein synthesis is not required for internalization. Host-cell actin polymerization was essential for the internalization of S. sputigena . Electron microscopy revealed that intracellular S. sputigena is located within a single membrane vesicle tightly wrapped around the bacterium which is decorated with LAMP1, indicating that S. sputigena is ultimately trafficked to a lysosome-like vesicle. Although S. sputigena mRNA was detectable within GECs, it rapidly decreased by 24 h post-inoculation. This differed from bacterial morphology, which remained intact up to 48 h. Induction of CXCL8 expression was concurrent with intracellular S. sputigena morphological integrity, suggesting that, while the bacteria are likely dead, bacterial macromolecules perpetuate inflammation in GECs. Collectively, this study demonstrates S. sputigena can be taken into gingival keratinocytes, where the bacterium induces inflammatory responses, but S. sputigena is ultimately processed by lysosomal machinery and is cleared from the intracellular niche in GECs.
Treponema denticola is a Gram-negative, anaerobic spirochete associated with periodontal disease. It navigates highly viscous environments using periplasmic flagella located between the inner and outer membranes. The flagellum is composed of three homologous flagellin proteins—FlaB1, FlaB2, and FlaB3—encoded by the flaB1 , flaB2 , and flaB3 genes, respectively, and is enwrapped by the sheath protein FlaA, encoded by the flaA gene. To investigate the roles of the three FlaB flagellins in cell morphology and motility, we constructed mutants lacking different combinations of the flaB genes, including double and triple deletions. The deletion of one or two flaB genes did not affect the transcription or protein expression of the remaining flaB gene(s). Normal flagellar filaments were observed when at least one FlaB was expressed but were absent in the complete flaB deletion mutant, which also exhibited an elongated cell shape. The flagella had no significant impact on bacterial growth. In liquid medium, most mutants exhibited rotational movement comparable to the parent strain, although some showed an increased population of cells with higher rotation rates. However, in a medium containing 0.5% agar, deletion of any two or three flaB genes significantly reduced motility. These findings suggest that the expression of at least two FlaB flagellins is required for the potent movement of T . denticola .
As first responders, neutrophils are a vital component of the host defense against oral pathogens, and their function is critical in preventing the progression of periodontal diseases. Streptococcus gordonii , a generally commensal oral bacterium, has been implicated in the pathogenesis of diseases by operating as a pathobiont with Porphyromonas gingivalis in periodontitis, and as an independent pathogen in infective endocarditis. Although the pathogenicity of S. gordonii is variable, its role in modulating, as well as responding to, host neutrophils remain, poorly understood. This study focuses on neutrophil activation, migration, and bactericidal activity towards S. gordonii . Our results found S. gordonii induced significant upregulation of surface markers CD63 and CD66 on neutrophils, a phenotypic change reminiscent of an oral neutrophil, and was enhanced by pre-activation of neutrophils by lipopolysaccharide (LPS) or the oral pathogen P. gingivalis . Co-incubations with P. gingivalis also led to a decreased ability of neutrophils to kill the normally commensal S. gordonii , though not other commensals with opportunistic pathogen potential, including Escherichia coli or Staphylococcus aureus . This increase in survival correlated with changes in phagosomal maturation, a decrease in cytoplasmic and phagosomal-associated granules, and increased IL-1β production. These results suggest oral streptococci may significantly contribute to oral neutrophil phenotypes associated with health, but introduction of oral pathogens can exacerbate a neutrophil shift and contribute to the persistence of S. gordonii , and its ability to contribute to the pathogenesis of periodontal disease.
Periodontal bacterial pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) accelerate inflammatory osteoclastogenesis, resulting in alveolar bone loss. The core PAMP and DAMP prototype molecules are periodontal bacterium Porphyromonas gingivalis-derived virulence lipids, for example, phosphoglycerol dihydroceramide (PGDHC) and lipopolysaccharide (LPS Pg), and the host non-histone alarmin high mobility group box protein-1 (HMGB1), respectively. Although it was reported that extracellularly released HMGB1 is critical for the promotion of sepsis inflammation in response to non-periodontal bacterial LPS, our understanding of the crosstalk between HMGB1 and P. gingivalis-derived virulence lipids remains limited. Therefore, we used Hmgb1fl/fl LysM-Cre+ mice with ablated HMGB1 mRNA and littermate Hmgb1fl/fl LysM-Cre- controls. We observed limited Hmgb1fl/fl LysM-Cre+ osteoclastogenesis compared to Hmgb1fl/fl in response to RANKL in vitro. Furthermore, recombinant HMGB1 protein restored osteoclast formation in Hmgb1fl/fl LysM-Cre+ cells, indicating the pivotal role of extracellular HMGB1 in osteoclastogenesis in vitro. Using bulk RNA-sequencing, we identified the diminished osteoclastogenesis in Hmgb1fl/fl LysM-Cre+ cells are linked to accelerated expression of canonical osteoclast-suppressing interferon genes. We surprisingly detected that PGDHC and LPS Pg accelerate osteoclastogenesis in Hmgb1fl/fl LysM-Cre+ cells in vitro. Using bulk RNA-sequencing and real-time PCR assays, we confirmed that PGDHC diminishes the expression patterns of different interferon-inducible guanylate-binding proteins (GBP 3, 4, 5, 9). At the same time, LPS Pg accelerates the expression of osteoclast-promoting matrix metalloproteases (MMP 8 and 12) mRNAs. The results suggest that the RANKL-primed osteoclastogenesis accelerated by P. gingivalis-derived virulence lipids is mediated by different MMP or GBP signaling pathways independently from canonical HMGB1 signaling.
Oral spirochetes are among the small group of keystone pathogens contributing to dysregulation of periodontal tissue homeostasis, leading to breakdown of the tissue and bone supporting the teeth in periodontal disease. Of the more than 60 oral Treponema species and phylotypes, Treponema denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is practicable. T. denticola is thus a model organism for studying spirochete behavior, metabolism, and interactions with other microbes and host tissues that are relevant to oral diseases. We recently demonstrated enhanced transformation efficiency using a synthetic shuttle plasmid resistant to T. denticola restriction-modification systems. Here, we report further optimization of the shuttle plasmid system by minimizing its size and by characterizing an array of promoter-gene constructs for plasmid-based genetic complementation, including the first inducible system for controlled expression of potentially toxic plasmid-encoded genes in Treponema. Our results highlight the importance of precise pairing of promoters and genes of interest for obtaining biologically optimal protein expression. This work expands the utility of the T. denticola shuttle plasmid system and will facilitate future studies in the analysis of Treponema physiology and behavior. Rigorous genetic analysis in oral spirochetes has been hampered by the limited utility of available versions of the Escherichia coli-T. denticola shuttle plasmid system. We report expanded characterization, refinement, and minimization of the shuttle plasmid, including relative activity of diverse promoters and the first inducible expression system described for T. denticola. We show that careful customization of the shuttle plasmid for specific applications is crucial for obtaining successful results.
Over the years, humanity has accumulated knowledge about the pathogens of infectious diseases and the ability of the human body to resist external aggression. In the last century, it became clear that the normal microflora of the human body can be used as an ally to resist a whole range of diseases. However, the intestinal microflora is the main object of modern complex studies. This review focuses on the microflora of the oral cavity. It describes the main microbiological composition of the microflora, including the most important bacterial species, fungi, and viruses. The main factors influencing the emergence of balance in the system “human oral cavity—microorganisms” are considered as well as environmental features that affect the formation of the species composition. The main functions performed by the oral microflora are described. Possible mechanisms for correcting initial dysbiotic disorders are also considered, including probiotics, bacteriophages, gases and thermotherapy, photobiomodulation, and diet correction.
Microbial infections and lipopolysaccharide (LPS)-induced senescence in human dental pulp cells (hDPCs) play a significant role in gingivitis etiology. However, the role of Apelin-12 in oral diseases, particularly its modulation of cellular senescence, remains poorly understood. This study investigated the protective effects of Apelin-12 against LPS-induced cellular senescence in hDPCs and its underlying mechanisms using cell isolation, culture, treatment, and transduction techniques, combined with reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, telomerase activity assays, senescence-associated β-galactosidase (SA-β-Gal) staining, and gene silencing. We first confirmed apelin receptor (APJ) expression in hDPCs and found that LPS significantly downregulated APJ at both mRNA and protein levels. Apelin-12 treatment restored telomerase activity and upregulated human telomerase reverse transcriptase (hTERT), while reducing senescence markers, including γH2AX and SA-β-Gal. Additionally, Apelin-12 suppressed the expression of senescence regulators p21 and acetylated p53 (ac-p53). Mechanistically, Apelin-12 restored SIRT6 (but not SIRT1) expression, and silencing SIRT6 abolished its anti-senescence effects, as evidenced by elevated p21, ac-p53, and SA-β-Gal, along with reduced hTERT and telomerase activity. These findings demonstrate that Apelin-12 attenuates LPS-induced cellular senescence in hDPCs via SIRT6-mediated pathways, suggesting its therapeutic potential for gingivitis management.
