Fusobacterium nucleatum exacerbates the progression of pulpitis by regulating the STING-dependent pathway.

Bacterial infection is the main cause of pulpitis. However, whether a dominant bacteria can promote the progression of pulpitis and its underlying mechanism remains unclear. We provided a comprehensive assessment of the microbiota alteration in pulpitis using 16S rRNA sequencing. Fusobacterium nucleatum was the most enriched in pulpitis and played a pathogenic role accelerating pulpitis progression in rat pulpitis model. After odontoblast-like cells cocultured with F. nucleatum, the stimulator of interferon genes (STING) pathway and autophagy were activation. There was a float of STING expression during F. nucleatum stimulation. STING was degraded by autophagy at the early stage. At the late stage, F. nucleatum stimulated mitochondrial Reactive Oxygen Species (ROS) production, mitochondrial dysfunction and then mtDNA escape into cytosol. mtDNA, which escaped into cytosol, caused more cytosolic mtDNA binds to cyclic GMP-AMP synthase (cGAS). The release of IFN-ß was dramatically reduced when mtDNA-cGAS-STING pathway inhibited. STING-/- mice showed milder periapical bone loss and lower serum IFN-ß levels compared with wildtype mice after 28 days F. nucleatum-infected pulpitis model establishment. Our data demonstrated that F. nucleatum exacerbated the progression of pulpitis, which was mediated by the STING-dependent pathway.

Potential role of epithelial-mesenchymal transition induced by periodontal pathogens in oral cancer.

With the increasing incidence of oral cancer in the world, it has become a hotspot to explore the pathogenesis and prevention of oral cancer. It has been proved there is a strong link between periodontal pathogens and oral cancer. However, the specific molecular and cellular pathogenic mechanisms remain to be further elucidated. Emerging evidence suggests that periodontal pathogens-induced epithelial-mesenchymal transition (EMT) is closely related to the progression of oral cancer. Cells undergoing EMT showed increased motility, aggressiveness and stemness, which provide a pro-tumour environment and promote malignant metastasis of oral cancer. Plenty of studies proposed periodontal pathogens promote carcinogenesis via EMT. In the current review, we discussed the association between the development of oral cancer and periodontal pathogens, and summarized various mechanisms of EMT caused by periodontal pathogens, which are supposed to play an important role in oral cancer, to provide targets for future research in the fight against oral cancer.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells.

Frankincense is produced by Boswellia trees, which can be found throughout the Middle East and parts of Africa and Asia. Boswellia serrata extract has been shown to have anti-cancer, anti-inflammatory, and antimicrobial effects. Periodontitis is an oral chronic inflammatory disease that affects nearly half of the US population. We investigated the antimicrobial effects of B. serrata extract on two oral pathogens associated with periodontitis. Using the minimum inhibitory concentration and crystal violet staining methods, we demonstrated that Porphyromonas gingivalis growth and biofilm formation were impaired by treatment with B. serrata extracts. However, the effects on Fusobacterium nucleatum growth and biofilm formation were not significant. Using quantification of colony-forming units and microscopy techniques, we also showed that concentrations of B. serrata that were not toxic for host cells decreased intracellular P. gingivalis infection in human gingival epithelial cells. Our results show antimicrobial activity of a natural product extracted from Boswellia trees (B. serrata) against periodontopathogens. Thus, B. serrata has the potential for preventing and/or treating periodontal diseases. Future studies will identify the molecular components of B. serrata extracts responsible for the beneficial effects.

Fusobacterium nucleatum, immune responses, and metastatic organ diversity in colorectal cancer liver metastasis.

The presence of Fusobacterium nucleatum is associated with an immunosuppressive tumor immune microenvironment (TIM) in primary colorectal cancer (CRC), contributing to tumor progression. Its persistence in CRC liver metastasis tissues raises questions about its role in modulating local and systemic immune responses and influencing recurrence patterns. This retrospective cohort study of 218 patients with CRC liver metastasis investigated the association of F. nucleatum in CRC liver metastasis tissues with systemic inflammation, TIM alterations, and the number of metastatic organs involved in recurrence. Two-step polymerase chain reaction (PCR), including digital PCR, detected F. nucleatum in 42% (92/218) of fresh-frozen specimens of CRC liver metastases. Compared with the F. nucleatum-none group, the F. nucleatum-high group showed higher C-reactive protein levels (0.82 vs. 0.22 mg/dL; Ptrend = 0.02), lower numbers of CD8+ cells (33.2 vs. 65.3 cells/mm2; Ptrend = 0.04) and FOXP3+ cells (11.3 vs. 21.7 cells/mm2; Ptrend = 0.01) in the TIM, and a greater number of metastatic organs involved in recurrence (1.6 vs. 1.1; p < 0.001). The presence of F. nucleatum in CRC liver metastasis tissues was associated with increased systemic inflammation, TIM alterations, and a greater number of metastatic organs involved in recurrence. These findings suggest a potential contribution of F. nucleatum to the metastatic propensity of CRC cells and could inform future research to enhance understanding of the interaction between tumor, host, and microbes in the metastatic process.

Fusobacterium nucleatum infection activates the noncanonical inflammasome and exacerbates inflammatory response in DSS-induced colitis.

Caspase activation results in pyroptosis, an inflammatory cell death that contributes to several inflammatory diseases by releasing inflammatory cytokines and cellular contents. Fusobacterium nucleatum is a periodontal pathogen frequently detected in human cancer and inflammatory bowel diseases. Studies have reported that F. nucleatum infection leads to NLRP3 activation and pyroptosis, but the precise activation process and disease association remain poorly understood. This study demonstrated that F. nucleatum infection exacerbates acute colitis in mice and activates pyroptosis through caspase-11-mediated gasdermin D cleavage in macrophages. Furthermore, F. nucleatum infection in colitis mice induces the enhancement of IL-1⍺ secretion from the colon, affecting weight loss and severe disease activities. Neutralization of IL-1⍺ protects F. nucleatum infected mice from severe colitis. Therefore, F. nucleatum infection facilitates inflammation in acute colitis with IL-1⍺ from colon tissue by activating noncanonical inflammasome through gasdermin D cleavage.

Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.

Gene inactivation by creating in-frame deletion mutations in Fusobacterium nucleatum is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX and radD, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes bamA and ftsZ, which are vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and Fusobacterium periodonticum. With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

Oral-gut microbial transmission promotes diabetic coronary heart disease.

BACKGROUND: Diabetes is a predominant driver of coronary artery disease worldwide. This study aims to unravel the distinct characteristics of oral and gut microbiota in diabetic coronary heart disease (DCHD). Simultaneously, we aim to establish a causal link between the diabetes-driven oral-gut microbiota axis and increased susceptibility to diabetic myocardial ischemia-reperfusion injury (MIRI). METHODS: We comprehensively investigated the microbial landscape in the oral and gut microbiota in DCHD using a discovery cohort (n = 183) and a validation chohort (n = 68). Systematically obtained oral (tongue-coating) and fecal specimens were subjected to metagenomic sequencing and qPCR analysis, respectively, to holistically characterize the microbial consortia. Next, we induced diabetic MIRI by administering streptozotocin to C57BL/6 mice and subsequently investigated the potential mechanisms of the oral-gut microbiota axis through antibiotic pre-treatment followed by gavage with specific bacterial strains (Fusobacterium nucleatum or fecal microbiota from DCHD patients) to C57BL/6 mice. RESULTS: Specific microbial signatures such as oral Fusobacterium nucleatum and gut Lactobacillus, Eubacterium, and Roseburia faecis, were identified as potential microbial biomarkers in DCHD. We further validated that oral Fusobacterium nucleatum and gut Lactobacillus are increased in DCHD patients, with a positive correlation between the two. Experimental evidence revealed that in hyperglycemic mice, augmented Fusobacterium nucleatum levels in the oral cavity were accompanied by an imbalance in the oral-gut axis, characterized by an increased coexistence of Fusobacterium nucleatum and Lactobacillus, along with elevated cardiac miRNA-21 and a greater extent of myocardial damage indicated by TTC, HE, TUNEL staining, all of which contributed to exacerbated MIRI. CONCLUSION: Our findings not only uncover dysregulation of the oral-gut microbiota axis in diabetes patients but also highlight the pivotal intermediary role of the increased abundance of oral F. nucleatum and gut Lactobacillus in exacerbating MIRI. Targeting the oral-gut microbiota axis emerges as a potent strategy for preventing and treating DCHD. Oral-gut microbial transmission constitutes an intermediate mechanism by which diabetes influences myocardial injury, offering new insights into preventing acute events in diabetic patients with coronary heart disease.

Extracellular vesicles from Aggregatibacter actinomycetemcomitans exhibit potential antitumorigenic effects in oral cancer: a comparative in vitro study.

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

Management of patient with Fusobacterim nucletum related pleural empyema: intrapleural antibiotic therapy can be considered for salvage therapy.

Pleural empyema can lead to significant morbidity and mortality despite chest drainage and antibiotic treatment, necessitating novel and minimally invasive interventions. Fusobacterium nucleatum is an obligate anaerobe found in the human oral and gut microbiota. Advances in sequencing and puncture techniques have made it common to detect anaerobic bacteria in empyema cases. In this report, we describe the case of a 65-year-old man with hypertension who presented with a left-sided encapsulated pleural effusion. Initial fluid analysis using metagenomic next-generation sequencing (mNGS) revealed the presence of Fusobacterium nucleatum and Aspergillus chevalieri. Unfortunately, the patient experienced worsening pleural effusion despite drainage and antimicrobial therapy. Ultimately, successful treatment was achieved through intrapleural metronidazole therapy in conjunction with systemic antibiotics. The present case showed that intrapleural antibiotic therapy is a promising measure for pleural empyema.

Fusobacterium nucleatum bacteremia complicated with intracranial Porphyromonas gingivalis and HSV-1 infection: a case report and literature review.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) belongs to the genus Fusobacterium, which is a gram-negative obligate anaerobic bacterium. Bacteremia associated with F. nucleatum is a serious complication, which is not common in clinic, especially when it is combined with other intracranial pathogenic microorganism infection. We reported for the first time a case of F. nucleatum bacteremia combined with intracranial Porphyromonas gingivalis (P. gingivalis) and herpes simplex virus type 1(HSV-1) infection. CASE PRESENTATION: A 60-year-old woman was admitted to our hospital with a headache for a week that worsened for 2 days. Combined with history, physical signs and examination, it was characterized as ischemic cerebrovascular disease (ICVD). F. nucleatum was detected in blood by matrix-assisted laser desorption/ionization time-offight mass spectrometry (MALDI-TOF-MS). Meanwhile, P. gingivalis and HSV-1 in cerebrospinal fluid (CSF) were identified by metagenome next generation sequencing (mNGS). After a quick diagnosis and a combination of antibiotics and antiviral treatment, the patient recovered and was discharged. CONCLUSION: To our knowledge, this is the first report of intracranial P. gingivalis and HSV-1 infection combined with F. nucleatum bacteremia.

Periodontal pathogen Fusobacterium nucleatum infection accelerates hepatic steatosis in high-fat diet-fed ApoE knockout mice by inhibiting Nrf2/Keap1 signaling.

AIMS: This study sought to explore the impact of Fusobacterium nucleatum on hepatic steatosis in apolipoprotein E (ApoE) knockout (KO) mice induced by a high-fat diet (HFD) and elucidate the underlying mechanism. METHODS: ApoE KO mice, on a HFD, received F. nucleatum oral inoculation every other day. After 24 weeks, body weight, liver weight, and liver index were assessed. Serum biochemistry and pro-inflammatory factors in serum and liver were analyzed. The histopathology of right maxilla and live were performed. Oil red O, immunohistochemistry, and immunofluorescence staining for the liver were conducted. Myeloperoxidase (MPO) activity, apoptosis, lipid reactive oxygen species (ROS), ROS, lipid peroxides, and hepatic lipids were also evaluated. Liver inflammation, fibrosis, de novo lipogenesis (DNL)-related molecule, and Nrf2/Keap1-related signaling molecule gene/protein expression were determined by real-time PCR (RT-PCR) and/or Western blot (WB) analysis. RESULTS: HFD-fed ApoE KO mice infected by F. nucleatum demonstrated significant changes, including increased body and liver weight, elevated proinflammatory factors and lipids in serum and liver, as well as neutrophil infiltration, fibrosis, apoptosis, oxidative stress, and lipid peroxidation in the liver. Additionally, F. nucleatum stimulates hepatic lipid accumulation and activates de novo lipogenesis (DNL), while simultaneously suppressing the Nrf2/Keap1 antioxidant pathway. CONCLUSION: In conclusion, our study reveals that oral inoculation of F. nucleatum might promote hepatic steatosis by inhibiting Nrf2/Keap1 pathway.

Autoinducer-2 produced by oral microbial flora and alveolar bone loss in periodontitis.

OBJECTIVE: The aim of this study was to investigate the association between autoinducer-2 (AI-2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI-2 may have the potential that monitor periodontitis and predict bone loss. BACKGROUND: Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. METHOD: We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI-2 in samples using the Vibrio harveyi BB180 bioluminescent-reporter system. To explore the interaction between AI-2 and bone metabolism, we utilized AI-2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI-2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi-species biofilm models to study the association between AI-2 and periodontal disease progression. RESULTS: The AI-2 concentration in GCF samples increased along with periodontal disease progression (p < .0001). F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner. In the periodontitis mice model, the CEJ-ABC distance in the F. nucleatum AI-2 treatment group was higher than that in the simple ligation group (p < .01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p < .05). CONCLUSIONS: We demonstrated that the AI-2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner and aggravated bone loss.

Cytokine responses of CD4+ T cells and NKT cells to periodontitis-associated bacteria in individuals with or without periodontitis.

AIM: Periodontitis is an inflammatory disease driven by opportunistic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum, where T-cell and NKT-cell responses to these bacteria in patients with periodontitis grade B or C are not fully elucidated. The objective is to determine if exaggerated proinflammatory Th-cell responses to periodontitis-associated bacteria, but not commensal bacteria, is a characteristic of increased periodontitis grade. METHODS: Mononuclear cells from patients with periodontitis grade C (n = 26) or grade B (n = 33) and healthy controls (HCs; n = 26) were stimulated with P. gingivalis, F. nucleatum or the commensal bacteria, Staphylococcus epidermidis and Cutibacterium acnes. Cytokine production by different T-cell populations and FOXP3-expression by regulatory T cells were assessed by flow cytometry. RESULTS: Compared to HCs, grade C patients had decreased frequencies of interleukin (IL)-10-producing CD4+ T cells before stimulation (p = .02) and increased frequencies of IFN-y-producing CD4+ T cells after stimulation with P. gingivalis (p = .0019). Grade B patients had decreased frequencies of FOXP3+ CD4+ T cells before (p = .030) before and after stimulation with anti-CD2/anti-CD3/anti-CD28-loaded beads (p = .047), P. gingivalis (p = .013) and S. epidermidis (p = .018). Clinical attachment loss correlated with the frequencies of IFN-y-producing Th1 cells in P. gingivalis- and F. nucleatum-stimulated cultures in grade B patients (p = .023 and p = .048, respectively) and with the frequencies of Th17 cells in P. gingivalis-stimulated cultures (p = .0062) in grade C patients. Patients with periodontitis grade C or grade B showed lower frequencies of IL-10-producing NKT cells than HCs in unstimulated cultures (p = .0043 and p = .027 respectively). CONCLUSIONS: Both periodontitis groups showed decreased frequencies of immunoregulatory T-cell and NKT cell subsets at baseline. Clinical attachment loss correlated with P. gingivalis-induced Th17-responses in grade C patients and with Th1-responses in grade B patients when cells were stimulated with P. gingivalis, supporting that dysregulated pro-inflammatory T-cell responses to periodontitis-associated bacteria contribute to the pathogenesis of periodontitis.

Unveiling the Hidden Links: Periodontal Disease, Fusobacterium Nucleatum, and Cancers.

PURPOSE OF REVIEW: Fusobacterium nucleatum (F. nucleatum), an anaerobic, gram-negative microbe, commonly found in human dental biofilm and the gut flora. It has long been known to have a higher concentration in periodontal disease and has recently been implicated in both oral and distant cancers such as colorectal, gastrointestinal, esophageal, breast, pancreatic hepatocellular, and genitourinary cancers. However, the mechanism of its involvement in the development of cancer has not been fully discussed. This review aims to cover biological molecular and clinical aspects of F. nucleatum and cancers. RECENT FINDINGS: Studies indicate F. nucleatum promotes tumor development through chronic inflammation, immune evasion, cell proliferation activation, and direct cell interactions, as in oral squamous cell carcinoma (OSCC). In colorectal cancer (CRC), F. nucleatum contributes to tumorigenesis through ß-catenin signaling and NF-κB activation. It also induces autophagy, leading to chemoresistance in CRC and esophageal cancers, and enhances tumor growth and metastasis in breast cancer by reducing T-cell infiltration. F. nucleatum is linked to carcinogenesis and increased bacterial diversity in OSCC, with improved oral hygiene potentially preventing OSCC. F. nucleatum triggers cancer by causing mutations and epigenetic changes through cytokines and reactive oxygen species. It also promotes chemoresistance in CRC. F. nucleatum may potentially serve as a diagnostic tool in various cancers, with non-invasive detection methods available. Further investigation is needed to discover its potential in the diagnosis and treatment of OSCC and other cancers.

Association of Fusobacterium nucleatum infection with the clinicopathological characteristics in colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) is a global health problem. The gut microbiome is now recognized as an important underlying factor to the initiation and progression of CRC. Fusobacterium nucleatum (FN) is one of the most studied bacteria in the aetiology of CRC. This study provided cohort evidence on the association of FN infection with clinicopathologic features in CRC patients. METHODS: We analysed the cancerous and adjacent non-cancerous formalin-fixed paraffin embedded (FFPE) tissue of 83 CRC patients from a single medical centre in Malaysia. TaqMan probe-based qPCR targeting the 16S rRNA gene was used to detect the presence of FN in the extracted FFPE DNA. The differences in FN expression between cancer and non-cancer tissues were evaluated. Association studies between FN infection in the tumour and relative FN abundance with available clinical data were conducted. RESULTS: FN was more abundant in the cancerous tissue compared to non-cancerous tissue (p = 0.0025). FN infection in the tumour was significantly associated with lymph node metastasis (p = 0.047) and cancer staging (p = 0.032), but not with other clinicopathologic variables. In double-positive patients where FN was detected in both cancerous and non-cancerous tissue, the expression fold-change of FN, calculated using 2-ΔΔCT formula, was significantly higher in patients with tumour size equal to or greater than 5 cm (p = 0.033) and in KRAS-mutated patients (p = 0.046). CONCLUSIONS: FN is enriched in CRC tumour tissue and is associated with tumour size, lymph node metastasis, cancer staging, and KRAS mutation in this single-centre small cohort study.

Coaggregated E. faecalis with F. nucleatum regulated environmental stress responses and inflammatory effects.

To investigate the cell-cell interactions of intergeneric bacterial species, the study detected the survival of Enterococcus faecalis (Ef) under monospecies or coaggregation state with Fusobacterium nucleatum subsp. polymorphum (Fnp) in environmental stress. Ef and Fnp infected the human macrophages with different forms (Ef and Fnp monospecies, Ef-Fnp coaggregates, Ef + Fnp cocultures) for exploring the immunoregulatory effects and the relevant molecular mechanisms. Meanwhile, the transcriptomic profiles of coaggregated Ef and Fnp were analyzed. Ef was shown to coaggregate with Fnp strongly in CAB within 90 min by forming multiplexes clumps. Coaggregation with Fnp reinforced Ef resistance against unfavorable conditions including alkaline, hypertonic, nutrient-starvation, and antibiotic challenges. Compared with monospecies and coculture species, the coaggregation of Ef and Fnp significantly facilitates both species to invade dTHP-1 cells and aid Ef to survive within the cells. Compared with coculture species, dual-species interaction of Ef and Fnp significantly decreased the levels of pro-inflammatory cytokines IL-6, TNF-α, and chemokines MCP-1 secreted by dTHP-1 cells and lessened the phosphorylation of p38, JNK, and p65 signaling pathways. The transcriptome sequencing results showed that 111 genes were differentially expressed or Ef-Fnp coaggregated species compared to Ef monospecies; 651 genes were differentially expressed for Fnp when coaggregation with Ef. The analysis of KEGG pathway showed that Ef differentially expressed genes (DEGs) were enriched in quorum sensing and arginine biosynthesis pathway; Fnp DEGs were differentially concentrated in lipopolysaccharide (LPS) biosynthesis, biofilm formation, and lysine degradation pathway compared to monospecies. KEY POINTS: • Coaggregated with Fnp aids Ef's survival in environmental stress, especially in root canals after endodontic treatment. • The coaggregation of Ef and Fnp may weaken the pro-inflammatory response and facilitate Ef to evade killed by macrophages. • The coaggregation between Ef and Fnp altered interspecies transcriptional profiles.

Screening inhibitors against the Ef-Tu of Fusobacterium nucleatum: a docking, ADMET and PBPK assessment study.

The oral pathogen Fusobacterium nucleatum has recently been associated with an elevated risk of colorectal cancer (CRC), endometrial metastasis, chemoresistance, inflammation, metastasis, and DNA damage, along with several other diseases. This study aimed to explore the disruption of protein machinery of F. nucleatum via inhibition of elongation factor thermo unstable (Ef-Tu) protein, through natural products. No study on Ef-Tu inhibition by natural products or in Fusobacterium spp. exists till todate. Ef-Tu is an abundant specialized drug target in bacteria that varies from human Ef-Tu. Elfamycins target Ef-Tu and hence, Enacyloxin IIa was used to generate pharmacophore for virtual screening of three natural product libraries, Natural Product Activity and Species Source (NPASS) (n = 30000 molecules), Tibetan medicinal plant database (n = 54 molecules) and African medicinal plant database (n > 6000 molecules). Peptaibol Septocylindrin B (NPC141050), Hirtusneanoside, and ZINC95486259 were prioritized from these libraries as potential therapeutic candidates. ADMET profiling was done for safety assessment, physiological-based pharmacokinetic modeling in human and mouse for getting insight into drug interaction with body tissues and molecular dynamics was used to assess stability of the best hit NPC141050 (Septocylindrin B). Based on the promising results, we propose further in vitro, in vivo and pharmacokinetic testing on the lead Septocylindrin B, for possible translation into therapeutic interventions.

Exosomes secreted by Fusobacterium nucleatum-infected colon cancer cells transmit resistance to oxaliplatin and 5-FU by delivering hsa_circ_0004085.

BACKGROUND: A large number of Fusobacterium nucleatum (Fn) are present in colorectal cancer (CRC) tissues of patients who relapse after chemotherapy, and Fn has been reported to promote oxaliplatin and 5-FU chemoresistance in CRC. Pathogens such as bacteria and parasites stimulate exosome production in tumor cells, and the regulatory mechanism of exosomal circRNA in the transmission of oxaliplatin and 5-FU chemotherapy resistance in Fn-infected CRC remains unclear. METHODS: Hsa_circ_0004085 was screened by second-generation sequencing of CRC tissues. The correlation between hsa_circ_0004085 and patient clinical response to oxaliplatin/5-FU was analyzed. Exosome tracing experiments and live imaging systems were used to test the effect of Fn infection in CRC on the distribution of hsa_circ_0004085. Colony formation, ER tracking analysis and immunofluorescence were carried out to verify the regulatory effect of exosomes produced by Fn-infected CRC cells on chemotherapeutic resistance and ER stress. RNA pulldown, LC-MS/MS analysis and RIP were used to explore the regulatory mechanism of downstream target genes by hsa_circ_0004085. RESULTS: First, we screened out hsa_circ_0004085 with abnormally high expression in CRC clinical samples infected with Fn and found that patients with high expression of hsa_circ_0004085 in plasma had a poor clinical response to oxaliplatin/5-FU. Subsequently, the circular structure of hsa_circ_0004085 was identified. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes produced by Fn-infected CRC cells transferred hsa_circ_0004085 between cells and delivered oxaliplatin/5-FU resistance to recipient cells by relieving ER stress. Hsa_circ_0004085 enhanced the stability of GRP78 mRNA by binding to RRBP1 and promoted the nuclear translocation of ATF6p50 to relieve ER stress. CONCLUSIONS: Plasma levels of hsa_circ_0004085 are increased in colon cancer patients with intracellular Fn and are associated with a poor response to oxaliplatin/5-FU. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes secreted by Fn-infected CRC cells deliver hsa_circ_0004085 between cells. Hsa_circ_0004085 relieves ER stress in recipient cells by regulating GRP78 and ATF6p50, thereby delivering resistance to oxaliplatin and 5-FU.

Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum.

A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions-termed coaggregation-by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the ΔcarR mutant. Significantly, the native culture medium of ΔkamA or ΔkamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the ΔkamA and ΔcarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (ΔradD or ΔcarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.