A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

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.

About a Possible Impact of Endodontic Infections by Fusobacterium nucleatum or Porphyromonas gingivalis on Oral Carcinogenesis: A Literature Overview.

Periodontitis is linked to the onset and progression of oral squamous cell carcinoma (OSCC), an epidemiologically frequent and clinically aggressive malignancy. In this context, Fusobacterium (F.) nucleatum and Porphyromonas (P.) gingivalis, two bacteria that cause periodontitis, are found in OSCC tissues as well as in oral premalignant lesions, where they exert pro-tumorigenic activities. Since the two bacteria are present also in endodontic diseases, playing a role in their pathogenesis, here we analyze the literature searching for information on the impact that endodontic infection by P. gingivalis or F. nucleatum could have on cellular and molecular events involved in oral carcinogenesis. Results from the reviewed papers indicate that infection by P. gingivalis and/or F. nucleatum triggers the production of inflammatory cytokines and growth factors in dental pulp cells or periodontal cells, affecting the survival, proliferation, invasion, and differentiation of OSCC cells. In addition, the two bacteria and the cytokines they induce halt the differentiation and stimulate the proliferation and invasion of stem cells populating the dental pulp or the periodontium. Although most of the literature confutes the possibility that bacteria-induced endodontic inflammatory diseases could impact on oral carcinogenesis, the papers we have analyzed and discussed herein recommend further investigations on this topic.

The impact of the human microbiome in tumorigenesis, cancer progression, and biotherapeutic development.

BACKGROUND: Cancer impacts millions of lives globally each year, with approximately 10 million cancer-related deaths recorded worldwide in 2020. Mounting research has recognised the human microbiome as a key area of interest in the pathophysiology of various human diseases including cancer tumorigenesis, progression and in disease outcome. It is suggested that approximately 20% of human cancers may be linked to microbes. Certain residents of the human microbiome have been identified as potentially playing a role, including: Helicobacter pylori, Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis and Porphyromonas gingivalis. MAIN BODY: In this review, we explore the current evidence that indicate a link between the human microbiome and cancer. Microbiome compositional changes have been well documented in cancer patients. Furthermore, pathogenic microbes harbouring specific virulence factors have been implicated in driving the carcinogenic activity of various malignancies including colorectal, gastric and pancreatic cancer. The associated genetic mechanisms with possible roles in cancer will be outlined. It will be indicated which microbes have a potential direct link with cancer cell proliferation, tumorigenesis and disease progression. Recent studies have also linked certain microbial cytotoxins and probiotic strains to cancer cell death, suggesting their potential to target the tumour microenvironment given that cancer cells are integral to its composition. Studies pertaining to such cytotoxic activity have suggested the benefit of microbial therapies in oncological treatment regimes. It is also apparent that bacterial pathogenic protein products encoded for by certain loci may have potential as oncogenic therapeutic targets given their possible role in tumorigenesis. CONCLUSION: Research investigating the impact of the human microbiome in cancer has recently gathered pace. Vast amounts of evidence indicate the human microbiome as a potential player in tumorigenesis and progression. Promise in the development of cancer biomarkers and in targeted oncological therapies has also been demonstrated, although more studies are needed. Despite extensive in vitro and in vivo research, clinical studies involving large cohorts of human patients are lacking. The current literature suggests that further intensive research is necessary to validate both the role of the human microbiome in cancer, and the use of microbiome modification in cancer therapy.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

Molecular Mechanisms Leading from Periodontal Disease to Cancer.

Periodontitis is prevalent in half of the adult population and raises critical health concerns as it has been recently associated with an increased risk of cancer. While information about the topic remains somewhat scarce, a deeper understanding of the underlying mechanistic pathways promoting neoplasia in periodontitis patients is of fundamental importance. This manuscript presents the literature as well as a panel of tables and figures on the molecular mechanisms of Porphyromonas gingivalis and Fusobacterium nucleatum, two main oral pathogens in periodontitis pathology, involved in instigating tumorigenesis. We also present evidence for potential links between the RANKL-RANK signaling axis as well as circulating cytokines/leukocytes and carcinogenesis. Due to the nonconclusive data associating periodontitis and cancer reported in the case and cohort studies, we examine clinical trials relevant to the topic and summarize their outcome.

Oral infectious bacteria in dental plaque and saliva as risk factors in patients with esophageal cancer.

BACKGROUND: High levels of periodontopathic bacteria as well as Streptococcus anginosus were detected in cancer tissue from patients with esophageal cancer. An association between oral infectious bacteria and esophageal cancer has been reported. METHODS: Characteristics of the oral microbiota and periodontal conditions were studied as clinicopathologic factors in patients with esophageal cancer. The study included 61 patients with esophageal cancer and 62 matched individuals without any cancers. Samples of subgingival dental plaque and unstimulated saliva were collected to evaluate the prevalence and abundance of the following oral bacteria using a real-time polymerase chain reaction assay: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, and S. anginosus. RESULTS: In the cancer group, the prevalence of all bacteria, with the exception of F. nucleatum, in dental plaque; the prevalence of A. actinomycetemcomitans in saliva; the abundance of all bacteria, with the exception of F. nucleatum and P. intermedia, in dental plaque; and the abundance of A. actinomycetemcomitans and S. anginosus in saliva were significantly higher. Furthermore, a logistic regression analysis suggested that the prevalence of T. forsythia and S. anginosus in dental plaque and of A. actinomycetemcomitans in saliva, as well as a drinking habit, were associated with a high risk of esophageal cancer, with a high odds ratio. CONCLUSIONS: The current findings have potential implications for the early diagnosis of esophageal cancer.

Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a gut microbe implicated in gastrointestinal tumorigenesis. Predicting the chemotherapeutic response is critical to developing personalised therapeutic strategies for oesophageal cancer patients. The present study investigated the relationship between F. nucleatum and chemotherapeutic resistance in oesophageal squamous cell carcinoma (ESCC). METHODS: We examined the relationship between F. nucleatum and chemotherapy response in 120 ESCC resected specimens and 30 pre-treatment biopsy specimens. In vitro studies using ESCC cell lines and co-culture assays further uncovered the mechanism underlying chemotherapeutic resistance. RESULTS: ESCC patients with F. nucleatum infection displayed lesser chemotherapeutic response. The infiltration and subsistence of F. nucleatum in the ESCC cells were observed by transmission electron microscopy and laser scanning confocal microscopy. We also observed that F. nucleatum modulates the endogenous LC3 and ATG7 expression, as well as autophagosome formation to induce chemoresistance against 5-FU, CDDP, and Docetaxel. ATG7 knockdown resulted in reversal of F. nucleatum-induced chemoresistance. In addition, immunohistochemical studies confirmed the correlation between F. nucleatum infection and ATG7 expression in 284 ESCC specimens. CONCLUSIONS: F. nucleatum confers chemoresistance to ESCC cells by modulating autophagy. These findings suggest that targeting F. nucleatum, during chemotherapy, could result in variable therapeutic outcomes for ESCC patients.

The Role of Fusobacterium nucleatum in Colorectal Carcinogenesis.

Colorectal cancer (CRC) is one of the most frequent and deadly neoplasms worldwide. Genetic factors, lifestyle habits, and inflammation are important risk factors associated with CRC development. In recent years, growing evidence has supporting the significant role of the intestinal microbiome in CRC carcinogenesis. Disturbances in the healthy microbial balance, known as dysbiosis, are frequently observed in these patients. Pathogenic microorganisms that induce intestinal dysbiosis have become an important target to determine the role of bacterial infection in tumorigenesis. Interestingly, the presence of different bacterial strains, such as Fusobacterium nucleatum, has been detected in tissue and stool from patients with CRC and associated with substantial clinical and molecular features, as well as with patient therapy response. Therefore, understanding how the presence and levels of F. nucleatumstrains in the gut affect the risk of CRC onset and progression may inform suitable candidates for interventions focused on modulation of this bacteria. Here we review new insights into the role of gut microbiota in CRC carcinogenesis and the clinical utility of using the detection of F. nucleatum in different settings such as screening, prognosis, and microbiota modulation as a means to prevent cancer, augment therapies, and reduce adverse effects of treatment.

Fusobacterium nucleatum facilitates ulcerative colitis through activating IL-17F signaling to NF-κB via the upregulation of CARD3 expression.

Accumulating evidence links Fusobacterium nucleatum with ulcerative colitis (UC). The mechanism by which F. nucleatum promotes intestinal inflammation in UC remains poorly defined. Here, we first examined the abundance and impact of F. nucleatum on disease activity in UC tissues. Next, we isolated a strain of F. nucleatum from UC tissues and explored whether F. nucleatum aggravates the intestinal inflammatory response in vitro and in vivo. We also examined whether F. nucleatum infection involves the NF-κB or IL-17F signaling pathways. Our data showed that F. nucleatum was enriched in 51.78% of UC tissues and was correlated with the clinical course, clinical activity and refractory behavior of UC (p < 0.05). Furthermore, we demonstrated that F. nucleatum promoted intestinal epithelial damage and the expression of the inflammatory cytokines IL-1ß, Il-6, IL-17F and TNF-α. Mechanistically, F. nucleatum targeted caspase activation and recruitment domain 3 (CARD3) through NOD2 to activate the IL-17F/NF-κB pathway in vivo and in vitro. Thus, F. nucleatum orchestrates a molecular network involving CARD3 and IL-17F to control the UC process. Measuring and targeting F. nucleatum and its associated pathways will yield valuable insight into the prevention and treatment of UC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fusobacterium nucleatum impedes remission of colitis in a mouse model.

The role of Fusobacterium nucleatum, often associated with intestinal diseases, in the remission of dextran sulfate sodium (DSS)-induced colitis was investigated. Female mice were divided into groups DC (DSS control) and DF (DSS + F. nucleatum). F. nucleatum (1.0 × 1010 cfu/mouse/day) in phosphate-buffered saline (PBS) was orally given to DF, while DC had PBS only. All mice had DSS in drinking water. In Experiment 1, mice underwent 2 inflammation phases, an in-between recovery phase and had their disease activity indices (DAI) calculated. Experiment 2 was similarly conducted, except that mice were dissected 3 days postrecovery, and had blood and colonic mucosal samples collected. In Experiment 1, DF had significantly (P < .05) higher DAI than DC, during the recovery and 2nd inflammation phases. In Experiment 2, genus Bacteroides was significantly (P < .05) higher and family Lachnospiraceae significantly lower in cecal mucosa-associated microbiota of DF than in that of DC. We concluded that F. nucleatum can impede colitis remission.

Adherent-invasive Escherichia coli (AIEC): Cause or consequence of inflammation, dysbiosis, and rupture of cellular joints in patients with IBD?

There are many factors contributing to the development of gastrointestinal diseases, grouped into genetic, environmental, and lifestyle factors. In recent years attention has fallen on pathogens; in particular, Bacteroides fragilis, Fusobacterium nucleatum, Escherichia coli (E. coli) and Helicobacter pylori have been studied. Several points remain to be clarified, and above all, as regards the adherent-invasive E. coli strains of E. coli, one wonders if they are a cause or a consequence of the disease. In this review, we have tried to clarify some points by examining a series of recent publications regarding the involvement of the bacterium in the pathology, even if other studies are necessary.

Fusobacterium nucleatum and colorectal cancer: A mechanistic overview.

Colorectal cancer (CRC) is the third most prevalent cancer in the world. There are many risk factors involved in CRC. According to recent findings, the tumor microenvironment and feces samples of patients with CRC are enriched by Fusobacterium nucleatum. Thus, F. nucleatum is proposed as one of the risk factors in the initiation and progression of CRC. The most important mechanisms of Fusobacterium nucleatum involved in CRC carcinogenesis are immune modulation (such as increasing myeloid-derived suppressor cells and inhibitory receptors of natural killer cells), virulence factors (such as FadA and Fap2), microRNAs (such as miR-21), and bacteria metabolism. The aim of this review was to evaluate the mechanisms underlying the action of F. nucleatum in CRC.

The Periodontopathic Bacterium Fusobacterium nucleatum Induced Proinflammatory Cytokine Production by Human Respiratory Epithelial Cell Lines and in the Lower Respiratory Organs in Mice.

BACKGROUND/AIMS: The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. METHODS: Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. RESULTS: Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. CONCLUSION: Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.

Role of NOD1/NOD2 receptors in Fusobacterium nucleatum mediated NETosis.

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.

Porphyromonas gingivalis induces depression via downregulating p75NTR-mediated BDNF maturation in astrocytes.

Many cross-sectional epidemiological studies have shown the incidence of periodontitis is positive correlated with that of depression. However, their causal relationship and underlying mechanism are largely unknown. Porphyromonas gingivalis (Pg) is the main pathogen for periodontitis. Employing female mice treated with Pg every other day for 4 weeks, we found that Pg-mice showed obvious depression-like behavior, an increased number of activated astrocytes and decreased levels of mature brain derived neurotrophic factor (BDNF) and astrocytic p75NTR in the hippocampus. Both hippocampal injection of BDNF and overexpression of p75NTR in astrocytes alleviated Pg-induced depression-like behavior in mice. Moreover, Pg-lipopolysaccharides (LPS) generated similar phenotypes, which were reversed by the TLR-4 inhibitor TAK242. Our results suggest that Pg-LPS decreases the level of astrocytic p75NTR and then downregulates BDNF maturation, leading to depression-like behavior in mice. Our study provides the first evidence that Pg is a modifiable risk factor for depression and uncovers a novel therapeutic target for the treatment of depression.

Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H2S and l-serine from l-cysteine and H2O, an unusual cysteine (hydroxyl) lyase reaction (ß-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp232 and Ser74, and the substrate α-carboxylate interacts with Thr73 and Gln147 Asp232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of ß-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser74, Thr73, and Gln147) exhibited reduced ß-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

Immunological Pathways Triggered by Porphyromonas gingivalis and Fusobacterium nucleatum: Therapeutic Possibilities?

Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are Gram-negative anaerobic bacteria possessing several virulence factors that make them potential pathogens associated with periodontal disease. Periodontal diseases are chronic inflammatory diseases of the oral cavity, including gingivitis and periodontitis. Periodontitis can lead to tooth loss and is considered one of the most prevalent diseases worldwide. P. gingivalis and F. nucleatum possess virulence factors that allow them to survive in hostile environments by selectively modulating the host's immune-inflammatory response, thereby creating major challenges to host cell survival. Studies have demonstrated that bacterial infection and the host immune responses are involved in the induction of periodontitis. The NLRP3 inflammasome and its effector molecules (IL-1ß and caspase-1) play roles in the development of periodontitis. We and others have reported that the purinergic P2X7 receptor plays a role in the modulation of periodontal disease and intracellular pathogen control. Caspase-4/5 (in humans) and caspase-11 (in mice) are important effectors for combating bacterial pathogens via mediation of cell death and IL-1ß release. The exact molecular events of the host's response to these bacteria are not fully understood. Here, we review innate and adaptive immune responses induced by P. gingivalis and F. nucleatum infections and discuss the possibility of manipulations of the immune response as therapeutic strategies. Given the global burden of periodontitis, it is important to develop therapeutic targets for the prophylaxis of periodontopathogen infections.

Bacterial species associated with persistent apical periodontitis exert differential effects on osteogenic differentiation.

AIM: To determine if bacteria associated with persistent apical periodontitis induce species-specific pro-inflammatory cytokine responses in macrophages, and the effects of this species-specific microenvironment on osteogenic differentiation. METHODOLOGY: Macrophages were exposed to Enterococcus faecalis, Streptococcus oralis, Streptococcus mitis, Fusobacterium nucleatum, Treponema denticola or Tannerella forsythia, and levels of TNF-α and IL-1ß elicited were determined by immunoassay. Following treatment of MG-63 pre-osteoblasts with conditioned media from bacteria-exposed macrophages, osteogenic differentiation and viability of osteoblasts were analyzed by Alizarin Red Staining and MTS assay, respectively. Statistical analysis was carried out by one-way anova with the Tukey post-hoc test. Differences were considered to be significant if P < 0.05. RESULTS: Macrophages exposed to Gram-positive bacteria did not produce significant amounts of cytokines. F. nucleatum-challenged macrophages produced up to four-fold more TNF-α and IL-1ß compared to T. denticola or T. forsythia. Only conditioned media from macrophages treated with Gram-negative bacteria decreased mineralization and viability of osteoblasts. CONCLUSIONS: Gram-positive bacteria did not impact osteogenic differentiation and appeared innocuous. Gram-negative bacteria, in particular F. nucleatum elicited an enhanced pro-inflammatory response in macrophages, inhibited osteogenic differentiation and reduced cell viability. The findings suggest that the presence of this organism could potentially increase the severity of persistent apical periodontitis.