Lavandula angustifolia Essential Oil Inhibits the Ability of Fusobacterium nucleatum to Produce Volatile Sulfide Compounds, a Key Components in Oral Malodor.

Oral malodor still constitutes a major challenge worldwide. A strong effort is invested in eliminating volatile sulfur compound-producing oral bacteria through organic natural products such as essential oils. Fusobacterium nucleatum is a known volatile sulfur compound-producing bacteria that inspires oral malodor. The aim of the present study was to test the effect of lavender essential oil on the bacterium's ability to produce volatile sulfide compounds, the principal components of oral malodor. Lavender (Lavandula angustifolia) essential oil was extracted by hydrodistillation and analyzed using GC-MS. The minimal inhibitory concentration (MIC) of lavender essential oil on Fusobacterium nucleatum was determined in a previous trial. Fusobacterium nucleatum was incubated anaerobically in the presence of sub-MIC, MIC, and above MIC concentrations of lavender essential oil, as well as saline and chlorhexidine as negative and positive controls, respectively. Following incubation, volatile sulfur compound levels were measured using GC (Oralchroma), and bacterial cell membrane damage was studied using fluorescence microscopy. Chemical analysis of lavender essential oil yielded five main components, with camphor being the most abundant, accounting for nearly one-third of the total lavender essential oil volume. The MIC (4 µL/mL) of lavender essential oil reduced volatile sulfur compound secretion at a statistically significant level compared to the control (saline). Furthermore, the level of volatile sulfur compound production attributed to 1 MIC of lavender essential oil was in the range of the positive control chlorhexidine with no significant difference. When examining bacterial membrane damage, 2 MIC of lavender essential oil (i.e., 8 µL/mL) demonstrated the same, showing antibacterial membrane damage values comparative to chlorhexidine. Since lavender essential oil was found to be highly effective in hindering volatile sulfur compound production by Fusobacterium nucleatum through the induction of bacterial cell membrane damage, the results suggest that lavender essential oil may be a suitable alternative to conventional chemical-based anti-malodor agents.

A gene delivery system with autophagy blockade for enhanced anti-angiogenic therapy against Fusobacterium nucleatum-associated colorectal cancer.

Anti-angiogenesis has emerged a promising strategy against colorectal cancer (CRC). However, the efficacy of anti-angiogenic therapy is greatly compromised by the up-regulated autophagy levels resulting from the evolutionary resistance mechanism and the presence of Fusobacterium nucleatum (F. nucleatum) in CRC. Herein, we report a cationic polymer capable of blocking autophagic flux to deliver plasmid DNA (pDNA) encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) for enhanced anti-angiogenic therapy against F. nucleatum-associated CRC. The autophagy-inhibiting cationic polymer, referred to as PNHCQ, is synthesized by conjugating hydroxychloroquine (HCQ) into 3,3'-diaminodipropylamine-pendant poly(ß-benzyl-L-aspartate) (PAsp(Nors)), which can be assembled and electrostatically interacted with sFlt-1 plasmid to form PNHCQ/sFlt-1 polyplexes. Hydrophobic HCQ modification not only boosts transfection efficiency but confers autophagy inhibition activity to the polymer. Hyaluronic acid (HA) coating is further introduced to afford PNHCQ/sFlt-1@HA for improved tumor targeting without compromising on transfection. Consequently, PNHCQ/sFlt-1@HA demonstrates significant anti-tumor efficacy in F. nucleatum-colocalized HT29 mouse xenograft model by simultaneously exerting anti-angiogenic effects through sFlt-1 expression and down-regulating autophagy levels exacerbated by F. nucleatum challenge. The combination of anti-angiogenic gene delivery and overall autophagy blockade effectively sensitizes CRC tumors to anti-angiogenesis, providing an innovative approach for enhanced anti-angiogenic therapy against F. nucleatum-resident CRC. STATEMENT OF SIGNIFICANCE: Up-regulated autophagy level within tumors is considered responsible for the impaired efficacy of clinic antiangiogenic therapy against CRC colonized with pathogenic F. nucleatum. To tackle this problem, an autophagy-inhibiting cationic polymer is developed to enable efficient intracellular delivery of plasmid DNA encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) and enhance anti-angiogenic therapy against F. nucleatum-associated CRC. HA coating that can be degraded by tumor-enriching hyaluronidase is further introduced for improved tumor targeting without compromising transfection efficiency. The well-orchestrated polyplexes achieve considerable tumor accumulation, efficient in vivo transfection, and effectively reinforce the sensitivity of CRC to the sFlt-1-derived anti-angiogenic effects by significantly blocking overall autophagy flux exacerbated by F. nucleatum challenge, thus harvesting robust antitumor outcomes against F. nucleatum-resident CRC.

Multimodal inhibitory effect of matcha on Porphyromonas gingivalis.

Porphyromonas gingivalis has been associated with progression of periodontitis, characterized by inflammation and destruction of periodontal tissues. Here, we report that matcha, a product of Camellia sinensis, hampers the adherence and survival of P. gingivalis through multiple tactics. Matcha extract (ME) inhibited the growth not only of P. gingivalis but also of Prevotella nigrescens and Fusobacterium nucleatum, while it did not inhibit growth of nine species of oral streptococci and Aggregatibacter actinomycetemcomitans. ME-mediated P. gingivalis growth inhibition was characterized by both morphological and physiological changes at the bacterial envelope, which were accompanied by nano-particle formation and decreased membrane fluidity/permeability without loss of membrane integrity. ME also triggered autoaggregation of P. gingivalis in a major fimbriae (FimA)-dependent manner. In addition, adherence of P. gingivalis was dramatically inhibited by ME, irrespective of fimbriae. Furthermore, a structure-activity relationship study tested a series of catechins isolated from ME and identified the pyrogallol-type B-ring of catechins as essential for P. gingivalis growth inhibition. In a clinical study to assess the microbiological and therapeutic effects of matcha mouthwash in patients with periodontitis, the P. gingivalis number in saliva was significantly reduced by matcha mouthwash compared to the pre-intervention level. A tendency toward improvement in probing pocket depth was observed in the matcha group, although the difference was not statistically significant. Taken together, we present a proof of concept, based on the multimodal inhibitory effect of matcha against P. gingivalis, and that matcha may have clinical applicability for prevention and treatment of periodontitis. IMPORTANCE: Periodontitis, a multifactorial inflammatory disease of the oral cavity, results in alveolar bone destruction, and is a major cause of tooth loss of humans. In addition, emerging evidence has demonstrated associations between periodontitis and a wide range of other chronic inflammation-driven disorders, including diabetes mellitus, preterm birth, cardiovascular disease, aspiration pneumonia, rheumatoid arthritis, cognitive disorder, and cancer. In the present study, we report that matcha, a product of Camellia sinensis, hampers Porphyromonas gingivalis, a major periodontal pathobiont, in not only a series of in vitro experiments but also a pilot intervention clinical trial of patients with periodontitis, in which matcha mouthwash statistically significantly reduced the P. gingivalis number in saliva, as compared to the pre-intervention level. Taken together, we suggest that matcha may have clinical applicability for prevention and treatment of periodontitis.

Efficacy of a mouthwash containing ε-poly-L-lysine, funme peptides and domiphen in reducing halitosis and supragingival plaque: a randomized clinical trial.

OBJECTIVE: To evaluate the antibacterial effectiveness of a combination of ε-poly-L-lysine (ε-PL), funme peptide (FP) as well as domiphen against oral pathogens, and assess the efficacy of a BOP® mouthwash supplemented with this combination in reducing halitosis and supragingival plaque in a clinical trial. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans were determined by the gradient dilution method. Subsequently, the CCK-8 assay was used to detect the toxicity of mouthwash on human gingival fibroblastst, and the effectiveness in reducing halitosis and supragingival plaque of the mouthwash supplemented with the combination was analyzed by a randomized, double-blind, parallel-controlled clinical trial. RESULTS: The combination exhibited significant inhibitory effects on tested oral pathogens with the MIC < 1.56% (v/v) and the MBC < 3.13% (v/v), and the mouthwash containing this combination did not inhibit the viability of human gingival fibroblasts at the test concentrations. The clinical trial showed that the test group displayed notably lower volatile sulfur compounds (VSCs) at 0, 10, 24 h, and 7 d post-mouthwash (P < 0.05), compared with the baseline. After 7 days, the VSC levels of the and control groups were reduced by 50.27% and 32.12%, respectively, and notably cutting severe halitosis by 57.03% in the test group. Additionally, the Plaque Index (PLI) of the test and control group decreased by 54.55% and 8.38%, respectively, and there was a significant difference in PLI between the two groups after 7 days (P < 0.01). CONCLUSIONS: The combination of ε-PL, FP and domiphen demonstrated potent inhibitory and bactericidal effects against the tested oral pathogens, and the newly formulated mouthwash added with the combination exhibited anti-dental plaque and anti-halitosis properties in a clinical trial and was safe. TRIAL REGISTRATION: The randomized controlled clinical trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023).

Norepinephrine may promote the progression of Fusobacterium nucleatum related colorectal cancer via quorum sensing signalling.

Fusobacterium nucleatum (F. nucleatum) is closely correlated with tumorigenesis in colorectal cancer (CRC). We aimed to investigate the effects of host norepinephrine on the carcinogenicity of F. nucleatum in CRC and reveal the underlying mechanism. The results revealed that both norepinephrine and bacterial quorum sensing (QS) molecule auto-inducer-2 (AI-2) were positively associated with the progression of F. nucleatum related CRC (p < 0.01). In vitro studies, norepinephrine induced upregulation of QS-associated genes and promoted the virulence and proliferation of F. nucleatum. Moreover, chronic stress significantly increased the colon tumour burden of ApcMin/+ mice infected with F. nucleatum (p < 0.01), which was decreased by a catecholamine inhibitor (p < 0.001). Our findings suggest that stress-induced norepinephrine may promote the progression of F. nucleatum related CRC via bacterial QS signalling. These preliminary data provide a novel strategy for the management of pathogenic bacteria by targeting host hormones-bacterial QS inter-kingdom signalling.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Engineering lauric acid-based nanodrug delivery systems for restoring chemosensitivity and improving biocompatibility of 5-FU and OxPt against Fn-associated colorectal tumor.

Colorectal cancer (CRC) occurs in the colorectum and ranks second in the global incidence of all cancers, accounting for one of the highest mortalities. Although the combination chemotherapy regimen of 5-fluorouracil (5-FU) and platinum(IV) oxaliplatin prodrug (OxPt) is an effective strategy for CRC treatment in clinical practice, chemotherapy resistance caused by tumor-resided Fusobacterium nucleatum (Fn) could result in treatment failure. To enhance the efficacy and improve the biocompatibility of combination chemotherapy, we developed an antibacterial-based nanodrug delivery system for Fn-associated CRC treatment. A tumor microenvironment-activated nanomedicine 5-FU-LA@PPL was constructed by the self-assembly of chemotherapeutic drug derivatives 5-FU-LA and polymeric drug carrier PPL. PPL is prepared by conjugating lauric acid (LA) and OxPt to hyperbranched polyglycidyl ether. In principle, LA is used to selectively combat Fn, inhibit autophagy in CRC cells, restore chemosensitivity of 5-FU as well as OxPt, and consequently enhance the combination chemotherapy effects for Fn-associated drug-resistant colorectal tumor. Both in vitro and in vivo studies exhibited that the tailored nanomedicine possessed efficient antibacterial and anti-tumor activities with improved biocompatibility and reduced non-specific toxicity. Hence, this novel anti-tumor strategy has great potential in the combination chemotherapy of CRC, which suggests a clinically relevant valuable option for bacteria-associated drug-resistant cancers.

A dendritic polyamidoamine supramolecular system composed of pillar[5]arene and azobenzene for targeting drug-resistant colon cancer.

Fusobacterium nucleatum caused drug-resistant around tumor sites often leads to the failure of chemotherapy during colorectal cancer (CRC) treatment. Multifunctional cationic quaternary ammonium materials have been widely used as broad-spectrum antibacterial agents in antibacterial and anticancer fields. Herein, we design a smart supramolecular quaternary ammonium nanoparticle, namely quaternary ammonium PAMAM-AZO@CP[5]A (Q-P-A@CP[5]A), consisting of azobenzene (AZO)-conjugated dendritic cationic quaternary ammonium polyamidoamine (PAMAM) as the core and carboxylatopillar[5]arene (CP[5]A)-based switch, for antibacterial and anti-CRC therapies. The quaternary ammonium-PAMAM-AZO (Q-P-A) core endows the supramolecular system with enhanced antibacterial and anticancer properties. -N+CH3 groups on the surface of Q-P-A are accommodated in the CP[5]A cavity under normal conditions, which significantly improves the biocompatibility of Q-P-A@CP[5]A. Meanwhile, the CP[5]A host can be detached from -N+CH3 groups under pathological conditions, achieving efficient antibacterial and antitumor therapies. Furthermore, azoreductase in the tumor site can break the -NN- bonds of AZO in Q-P-A@CP[5]A, leading to the morphology recovery of supramolecular nanoparticles and CRC therapy through inducing cell membrane rupture. Both in vitro and in vivo experiments demonstrate that Q-P-A@CP[5]A possesses good biocompatibility, excellent antibacterial effect, and CRC treatment capability with negligible side effects. This supramolecular quaternary ammonium system provides an effective treatment method to overcome chemotherapy-resistant cancer caused by bacteria.

Effect of cranberry juice deacidification on its antibacterial activity against periodontal pathogens and its anti-inflammatory properties in an oral epithelial cell model.

Cranberries are widely recognized as a functional food that can promote oral health. However, the high concentration of organic acids in cranberry juice can cause tooth enamel erosion. Electrodialysis with bipolar membrane (EDBM) is a process used for the deacidification of cranberry juice. The present study investigated whether the removal of organic acids (0%, 19%, 42%, 60%, and 79%) from cranberry juice by EDBM affects its antibacterial activity against major periodontopathogens as well as its anti-inflammatory properties in an oral epithelial cell model. A deacidification rate ≥60% attenuated the bactericidal effect against planktonic and biofilm-embedded Aggregatibacter actinomycetemcomitans but had no impact on Porphyromonas gingivalis and Fusobacterium nucleatum. Cranberry juice increased the adherence of A. actinomycetemcomitans and P. gingivalis to oral epithelial cells, but reduced the adherence of F. nucleatum by half regardless of the deacidification rate. F. nucleatum produced more hydrogen sulfide when it was exposed to deacidified cranberry juice with a deacidification rate ≥42% compared to the raw beverage. Interestingly, the removal of organic acids from cranberry juice lowered the cytotoxicity of the beverage for oral epithelial cells. Deacidification attenuated the anti-inflammatory effect of cranberry juice in an in vitro oral epithelial cell model. The secretion of IL-6 by lipopolysaccharide (LPS)-stimulated oral epithelial cells exposed to cranberry juice increased proportionally with the deacidification rate. No such effect was observed with respect to the production of IL-8. This study provided evidence that organic acids, just like phenolic compounds, might contribute to the health benefits of cranberry juice against periodontitis.

Cytocompatibility and Synergy of EGCG and Cationic Peptides Against Bacteria Related to Endodontic Infections, in Planktonic and Biofilm Conditions.

This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of epigallocatechin-3-gallate (EGCG) associated with peptide LL-37 and its analogue KR-12-a5 against oral pathogens. The effect of the compounds on metabolism of fibroblasts was evaluated by methyltetrazolium assays. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on single- and dual-species biofilms and E. faecalis biofilms in dentinal tubules and analyzed by bacterial counts and confocal microscopy. Data were statistically analyzed considering p < 0.05. EGCG and peptide combinations were not toxic to fibroblasts. KR-12-a5 showed synergistic or addictive effects with EGCG and LL-37 against all bacteria tested. However, EGCG associated with KR-12-a5 demonstrated the highest bactericidal activity on all bacteria tested, at lower concentrations. In single-species biofilms, EGCG + KR-12-a5 eliminated S. mutans and A. israelii and reduced E. faecalis and F. nucleatum counts around 5 log CFU/mL. EGCG + KR-12-a5 reduced E. faecalis (-3.93 log CFU/mL) and eliminated S. mutans in dual-species biofilms. No growth of E. faecalis and significant reduction in A. israelii (-6.24 log CFU/mL) and F. nucleatum (-4.62 log CFU/mL) counts were detected in dual-species biofilms. The combination of EGCG and KR-12-a5 led to 88% of E. faecalis dead cells inside dentin tubules. The association of EGCG and KR-12-a5 was cytocompatible and promoted synergistic effect against biofilms of bacteria associated with endodontic infections.

Septic hip abscess due to Fusobacterium nucleatum and Actinomyces turicensis in an immunocompetent SARS-CoV-2 positive patient.

A 42-year-old man was referred to the Department of Orthopedic Surgery with pain over his right greater trochanter and signs of systemic infection. CT showed an enhanced mass in his gluteus maximus as well as gas in the biceps femoris over the underlying hip joint. Tissue biopsy yielded Fusobacterium nucleatum and Actinomyces turicensis. The patient was successfully treated for 6 weeks with amoxicillin/clavulanic acid 875mg/125mg and metronidazole 500mg.

A cocoa (Theobroma cacao L.) extract impairs the growth, virulence properties, and inflammatory potential of Fusobacterium nucleatum and improves oral epithelial barrier function.

Fusobacterium nucleatum is associated with many conditions and diseases, including periodontal diseases that affect tooth-supporting tissues. The aim of the present study was to investigate the effects of a cocoa extract (Theobroma cacao L.) on F. nucleatum with respect to growth, biofilm formation, adherence, and hydrogen sulfide (H2S) production. The anti-inflammatory properties and the effect on epithelial barrier function of the cocoa extract were also assessed. The cocoa extract, whose major phenolic compound is epicatechin, dose-dependently inhibited the growth, biofilm formation, adherence properties (basement membrane matrix, oral epithelial cells), and H2S production of F. nucleatum. It also decreased IL-6 and IL-8 production by F. nucleatum-stimulated oral epithelial cells and inhibited F. nucleatum-induced NF-κB activation in monocytes. Lastly, the cocoa extract enhanced the barrier function of an oral epithelial model by increasing the transepithelial electrical resistance. We provide evidence that the beneficial properties of an epicatechin-rich cocoa extract may be useful for preventing and/or treating periodontal diseases.

Inhibitory effect of black cumin (Nigella sativa) seed essential oil on Fusobacterium nucleatum L-methionine-γ-lyase (L-methioninase) activity.

The enzyme L-methionine-γ-lyase is commonly found in a wide range of bacteria and catalyzes the α-elimination and γ-elimination of L-methionine to produce methyl mercaptan, α-ketobutyrate and ammonia. Black cumin seed essential oil (BC oil) reportedly exhibits deodorizing activity against methyl mercaptan. Therefore, we hypothesized that BC oil may also suppress methyl mercaptan production. In this study, we aimed to evaluate the inhibitory effect of BC oil on L-methionine-γ-lyase activity in Fusobacterium nucleatum. Recombinant L-methionine-γ-lyase was incubated under appropriate conditions with BC oil and its constituent thymoquinone. To analyze L-methionine-γ-lyase activity, α-ketobutyric acid and ammonia concentrations were determined. The concentrations of α-ketobutyric acid and ammonia were significantly decreased by 10 µg mL-1 of BC oil (P < 0.01) and 16.4 µg/mL of thymoquinone (P < 0.05). An enzyme kinetic assay showed a mixed inhibition pattern between L-methionine-γ-lyase and thymoquinone. In conclusion, BC oil not only had a deodorizing effect against methyl mercaptan but also an inhibitory effect on methyl mercaptan production through the suppression of L-methionine-γ-lyase activity. Thymoquinone may be mainly responsible for these effects of BC oil. Thus, application of natural BC oil may be adapted not only for medical use but also in other areas of life.

Aspirin Modulation of the Colorectal Cancer-Associated Microbe Fusobacterium nucleatum.

Aspirin is a chemopreventive agent for colorectal adenoma and cancer (CRC) that, like many drugs inclusive of chemotherapeutics, has been investigated for its effects on bacterial growth and virulence gene expression. Given the evolving recognition of the roles for bacteria in CRC, in this work, we investigate the effects of aspirin with a focus on one oncomicrobe-Fusobacterium nucleatum We show that aspirin and its primary metabolite salicylic acid alter F. nucleatum strain Fn7-1 growth in culture and that aspirin can effectively kill both actively growing and stationary Fn7-1. We also demonstrate that, at levels that do not inhibit growth, aspirin influences Fn7-1 gene expression. To assess whether aspirin modulation of F. nucleatum may be relevant in vivo, we use the ApcMin/+ mouse intestinal tumor model in which Fn7-1 is orally inoculated daily to reveal that aspirin-supplemented chow is sufficient to inhibit F. nucleatum-potentiated colonic tumorigenesis. We expand our characterization of aspirin sensitivity across other F. nucleatum strains, including those isolated from human CRC tissues, as well as other CRC-associated microbes, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli Finally, we determine that individuals who use aspirin daily have lower fusobacterial abundance in colon adenoma tissues, as determined by quantitative PCR performed on adenoma DNA. Together, our data support that aspirin has direct antibiotic activity against F. nucleatum strains and suggest that consideration of the potential effects of aspirin on the microbiome holds promise in optimizing risk-benefit assessments for use of aspirin in CRC prevention and management.IMPORTANCE There is an increasing understanding of the clinical correlations and potential mechanistic roles of specific members of the gut and tumoral microbiota in colorectal cancer (CRC) initiation, progression, and survival. However, we have yet to parlay this knowledge into better CRC outcomes through microbially informed diagnostic, preventive, or therapeutic approaches. Here, we demonstrate that aspirin, an established CRC chemopreventive, exhibits specific effects on the CRC-associated Fusobacterium nucleatum in culture, an animal model of intestinal tumorigenesis, and in human colonic adenoma tissues. Our work proposes a potential role for aspirin in influencing CRC-associated bacteria to prevent colorectal adenomas and cancer, beyond aspirin's canonical anti-inflammatory role targeting host tissues. Future research, such as studies investigating the effects of aspirin on fusobacterial load in patients, will help further elucidate the prospect of using aspirin to modulate F. nucleatumin vivo for improving CRC outcomes.

Fusobacterium nucleatum Promotes Cisplatin-Resistance and Migration of Oral Squamous Carcinoma Cells by Up-Regulating Wnt5a-Mediated NFATc3 Expression.

Bacterial infection contributes to tumor development and malignant progression. Fusobacterium nucleatum (F. nucleatum) is reported to promote oral squamous cell carcinoma. However, molecular bases of F. nucleatum regulating oral cancer cells have not been fully elucidated. We report here that F. nucleatum down-regulates p53 and E-cadherin via the Wnt/NFAT pathway to promote cisplatin-resistance and migration in oral squamous carcinoma cells. We pretreated Cal-27 and HSC-3 cells with F. nucleatum and the survival rates against cysplatin (Cis-diamminedichloroplatinum, CDDP) were significantly higher in treated cells. The expressions of migration and apoptosis-related proteins like E-cadherin and p53 were lower in western blot analysis. We observed that F. nucleatum was an activator of the Wnt/NFAT pathway. The expression levels of the Wnt pathway gene wnt5a and Nuclear factors of activated T cells 3 (NFATc3) were notably higher in treated cells. With the inhibition effect of NFAT-inhibitory peptide VIVIT, the expressions of E-cadherin and p53 in response to F. nucleatum infection were up-regulated reversely. We concluded that F. nucleatum might promote cisplatin-resistance and migration of oral squamous cell carcinoma cells through the Wnt/NFAT pathway.

Heat-killed Lactobacillus acidophilus mediates Fusobacterium nucleatum induced pro-inflammatory responses in epithelial cells.

Probiotics is widespreadly used nowadays. However, the safety issue with the use of live probiotics is still a matter of contention. In recent years, an expanding body of evidence supports the beneficial role of heat-killed probiotics in the maintenance of systemic health, whereas the role of these heat-killed bacteria on periodontal health remains unclear. This study aimed to evaluate the effects of heat-killed probiotics on periodontal pathogen virulence and associated mechanisms. We demonstrated that heat-killed Lactobacillus acidophilus was able to coaggregate with Fusobacterium nucleatum, the bridging bacteria of oral biofilm, and inhibit the adhesion and invasion of F. nucleatum, leading to a subsequent elimination of pro-inflammatory cytokine production in oral epithelial cells. This coaggregation further caused a suppression of the virulence gene fap2 expression in F. nucleatum. Therefore, heat-killed L. acidophilus might downregulate the pro-inflammatory cytokine expression in epithelial cells via coaggregation with F. nucleatum and suppression of F. nucleatum fap2 expression, which was the first demonstration that heat-killed probiotics modulate periodontal disease pathogenesis via coaggregation. Collectively, this finding provides new evidence that heat-killed probiotics might exert beneficial effects to periodontal health by coaggregating with periodontal pathogens and modulating their virulence.

New Roles for Fusobacterium nucleatum in Cancer: Target the Bacteria, Host, or Both?

Fusobacterium nucleatum is an oral bacterium associated with colorectal cancer (CRC) proliferation, chemoresistance, inflammation, metastasis, and now DNA damage. While controlling F. nucleatum through antibiotics could reduce cancer severity, this article proposes additional strategies to block Fusobacterium-host interactions, as well as treatment of activated host immune and oncogenic signaling pathways in CRC.

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

Dimethylated acylphloroglucinol meroterpenoids with anti-oral-bacterial and anti-inflammatory activities from Hypericum elodeoides.

Acylphloroglucinol meroterpenoids are adducts of the acylphloroglucinol unit and polyprenylated fragments (terpenoids) with attractive structures and bioactivities. During study of the medicinal molecules of the genus Hypericum, the first example of dimethylated acylphloroglucinol meroterpenoids with pyran-fused 6/6/6 tricyclic skeletons ((+)/(-)-elodeoidols A-F (1-6)), along with three biogenetical homologues (7-9) were isolated from the herbaceous plant of Hypericum elodeoides. Their structures including absolute configurations were then identified by nuclear magnetic resonance (NMR), high resolution electrospray ionization mass spectroscopy (HRESIMS), electronic circular dichroism (ECD) analysis and calculations. The monoterpene moiety of 1-6 were cyclized as two cyclohexanes and fused with a dimethylated acylphloroglucinol unit through an additional ether linkage, which led to an interesting pyran-fused linear or angle type 6/6/6 tricyclic skeleton. Compounds 5, 8 and 9 showed preferable antibacterial activities against three oral bacteria, among the MIC value of (+)-5 was 6.25 µg/ml; Compounds 3, 7 and 8 exhibited significant NO inhibitory activity against LPS induced RAW264.7 cells (IC50: 10.39 ± 0.49 ~ 34.25 ± 2.32 µM).

Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.