Metagenomic Analysis of Bacterial Microflora in Dental and Atherosclerotic Plaques of Patients With Internal Carotid Artery Stenosis.

Background: Internal carotid artery stenosis is primarily attributed to atherosclerosis in the carotid artery bifurcation. Previous studies have detected oral bacteria in atherosclerotic lesions, suggesting an association between oral bacteria and atherosclerosis. In this study, we compared the bacterial flora of the atherosclerotic plaque in the carotid artery and dental plaque of patients with internal carotid artery stenosis using 16S ribosomal RNA (16S rRNA) metagenomic sequencing. Methods: Fifty-four patients who underwent internal carotid endarterectomy for internal carotid artery stenosis at the Showa University Hospital between April 2016 and February 2018 were included. Polymerase chain reaction targeting the 16S rRNA gene detected bacterial DNA in the carotid plaques of 11 cases, of which only 5 could be further analyzed. Thereafter, DNA extracted from the carotid and oral plaques of these 5 cases were analyzed using metagenomic sequencing targeting 16S rRNA. In addition, their general condition and oral conditions were evaluated. The patients were classified into symptomatic and asymptomatic groups based on the presence or absence of symptoms of transient ischemic attack, and their bacterial flora was evaluated. Results: The results demonstrated that the microflora of carotid plaques (n = 5) contained bacterial species from 55 families and 78 genera. In addition, 86.5% of the bacteria detected in the carotid plaques were also detected in oral plaques. Cariogenic and periodontopathic bacteria accounted for 27.7% and 4.7% of the bacteria in the carotid plaques, respectively. Conclusions: These results suggest that oral bacteria are directly or indirectly involved in the pathogenesis of atherosclerosis. More extensive studies of oral commensal bacteria detected in extra-oral lesions are warranted to comprehensively investigate the role of oral bacteria in the pathogenesis of systemic diseases.

The Klebsiella mannose phosphotransferase system promotes proliferation and the production of extracellular polymeric substances from mannose, facilitating adaptation to the host environment.

OBJECTIVES: Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. METHODS: We generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. RESULTS: We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. CONCLUSION: The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state.

OBJECTIVES: Commensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions. METHODS: We used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively. RESULTS: We found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils. CONCLUSIONS: The presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

Novel antibody assessment method for microbial compositional alteration in the oral cavity.

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.

Particle Size Analysis in Aerosol-Generating Dental Procedures Using Laser Diffraction Technique.

The global outbreak of coronavirus disease 2019 (COVID-19) has raised concerns about the risk of airborne infection during dental treatment. Aerosol-generating dental procedures (AGDP) produce droplets and aerosols, but the details of the risks of COVID-19 transmission in AGDP are not well-understood. By discriminating between droplets and aerosols, we devised a method to measure particle size using laser diffraction analysis and evaluated aerosols generated from dental devices for providing a basis for proper infection control procedures. The droplets and aerosols generated from dental devices were characterized by multimodal properties and a wide range of droplet sizes, with the majority of droplets larger than 50 µm. AGDP emitted few aerosols smaller than 5 µm, which are of concern for pulmonary infections due to airborne transmission. In addition, the use of extraoral suction was found to prevent the spread of aerosols from high-speed dental engines. This study suggests that the risk of aerosol infections is considerably limited in regular dental practice and that current standard precautions, such as mainly focusing on protection against droplet and contact infections, are sufficient. While several cases of airborne transmission of COVID-19 in general clinics and emergency hospitals have been reported, cluster outbreaks in dental clinics have not yet been reported, which may indicate that AGDP does not pose a significant threat in contributing to the spread of SARS-CoV-2.

Dysregulation of Intestinal Microbiota Elicited by Food Allergy Induces IgA-Mediated Oral Dysbiosis.

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

Streptococcus sanguinis Noncoding cia-Dependent Small RNAs Negatively Regulate Expression of Type IV Pilus Retraction ATPase PilT and Biofilm Formation.

Small noncoding RNAs (sRNAs) have been identified as important regulators of gene expression in various cellular processes. cia-dependent small RNAs (csRNAs), a group of sRNAs that are controlled by the two-component regulatory system CiaRH, are widely conserved in streptococci, but their targets have been identified only in Streptococcus pneumoniaeStreptococcus sanguinis, a pioneer colonizer of teeth and one of the most predominant bacteria in the early oral biofilm, has been shown to have six csRNAs. Using computational target prediction and the luciferase reporter assay, we identified pilT, a constituent of the type IV pilus operon, as a negative regulatory target for one of the csRNAs, namely, csRNA1-1, in S. sanguinis RNA-RNA electrophoretic mobility shift assay using a nucleotide exchange mutant of csRNA1-1 revealed that csRNA1-1 binds directly to pilT mRNA. In addition, csRNA1-1 and csRNA1-2, a putative gene duplication product of csRNA1-1 that is tandemly located in the S. sanguinis genome, negatively regulated S. sanguinis biofilm formation. These results suggest the involvement of csRNAs in the colonization step of S. sanguinis.

Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages.

Melatonin produced by the pineal gland suppresses inflammatory responses in innate immune cells. However, the mechanism of how melatonin affects inflammatory gene regulation remains unclear. Here we performed comprehensive microarray analysis combined with transcription factor binding site (TFBS) analysis using LPS-induced mouse macrophages to investigate the effect of melatonin treatment. The results showed that melatonin preferentially downregulated interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) related signaling. The results also showed that melatonin strongly suppressed virus infection related gene expression. Furthermore, TFBS analysis implicated that melatonin downregulated the binding activity of hypoxia inducible factors (HIFs), following destabilizing actin cytoskeleton which are indispensable for induction of the TRIF-dependent signaling pathway. Indeed, it was demonstrated that melatonin treatment caused impaired phagocytosis in macrophages. Thus, melatonin regulates inflammatory responses by inhibiting specific subsets of transcription factors (TFs) by disrupting actin dynamics in the macrophage.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide.

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Determination of reactive oxygen generated from natural medicines and their antibacterial activity.

Extracts of 16 natural medicine powders (Galla chinensis, Malloti cortex, Cassiae semen, Sophorae radix, Myricae cortex, Crataegi fructus, Gambir, Mume fructus, Geranii herba, Phellodendri cortex, Coptidis rhizoma, Swertiae herba, and Cinnamomi cortex) were assayed for reactive oxygen concentrations using the peroxyoxalate chemiluminescent detection system. High luminescence intensity was observed in Galla chinensis, Geranii herba, Malloti cortex, Myricae cortex, and Cinnamomi cortex. Additional experiments identified the reactive oxygen species as hydrogen peroxide. Galla chinensis generated 2.4×10-4 mol/L hydrogen peroxide from a 1 mg/mL solution. In bacterial growth tests, Galla chinensis extract had antibacterial activity against Escherichia coli, Staphylococcus aureus, Bacteroides thetaiotaomicron, Campylobacter sputorum biovar sputorum, Streptococcus salivarius thermophilus, Lactobacillus casei, and Bifidobacterium longum infantis. This antibacterial activity was decreased by the addition of catalase. It revealed that hydrogen peroxide which Galla chinensis produced participated in antibacterial activity.

Effects of Hangeshashinto on Growth of Oral Microorganisms.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.

Antibacterial activities of persimmon extracts relate with their hydrogen peroxide concentration.

Persimmon, a deciduous tree of the family Ebenaceae, is found throughout East Asia and contains high levels of tannins. This class of natural compounds exhibit favorable toxicity profiles along with bactericidal activity without the emergence of resistant bacteria, suggesting potential medical applications. Consistent with these observations, persimmon leaves show antibacterial activity. However, the mechanism of persimmon antibacterial activity remains unknown. In the present work, we demonstrate that the antibacterial activity of persimmon reflects the generation of reactive oxygen from tannins. The identification and quantification of reactive oxygen generated from persimmon and the level of antibacterial activity were determined.

Rothia dentocariosa induces TNF-alpha production in a TLR2-dependent manner.

Previous work suggested that Rothia dentocariosa is associated with periodontal inflammatory disease. However, little is known about the pathogenicity of this bacterium. To characterize host response to this bacterium, we measured (via ELISA) the amount of TNF-α in the culture supernatant following the stimulation of THP-1 cells (a human acute monocytic leukemia cell line) with R. dentocariosa cells (ATCC14189 and ATCC14190). Exposure to bacterial cells induced the production of TNF-α in a dose-dependent manner. The bacterial induction of TNF-α in THP-1 cells was mediated by the Toll-like receptor 2 (TLR2), as demonstrated by gene-specific knockdown via siRNA, which successfully suppressed TLR2 expression and significantly inhibited the production of TNF-α in the culture supernatant. To confirm the role of TLR2, we examined TLR2-dependent NF-κB activation by R. dentocariosa cells in a distinct cell line. Specifically, HEK293 cells were transiently cotransfected with the human TLR2 gene and an NF-κB-dependent luciferase-encoding reporter gene. The bacterial cells induced NF-κB activation in the transfected HEK293 cells in a dose-dependent manner. In contrast, bacterial cells failed to induce NF-κB activation in cells transfected with pEF6 control vector. Taken together, these results suggest that R. dentocariosa induces host TNF-α production by a TLR2-dependent mechanism.

Identification and functional analysis of the gene cluster for fructan utilization in Prevotella intermedia.

Fructanase enzymes hydrolyze the ß-2,6 and ß-2,1 linkages of levan and inulin fructans, respectively. We analyzed the influence of fructan on the growth of Prevotella intermedia. The growth of P. intermedia was enhanced by addition of inulin, implying that P. intermedia could also use inulin. Based on this finding, we identified and analyzed the genes encoding a putative fructanase (FruA), sugar transporter (FruB), and fructokinase (FruK) in the genome of strain ATCC25611. Transcript analysis by RT-PCR showed that the fruABK genes were co-transcribed as a single mRNA and semi-quantitative analysis confirmed that the fruA gene was induced in response to fructose and inulin. Recombinant FruA and FruK were purified and characterized biochemically. FruA strongly hydrolyzed inulin, with slight degradation of levan via an exo-type mechanism, revealing that FruA is an exo-ß-d-fructanase. FruK converted fructose to fructose-6-phosphate in the presence of ATP, confirming that FruK is an ATP-dependent fructokinase. These results suggest that P. intermedia can utilize fructan as a carbon source for growth, and that the fructanase, sugar transporter, and fructokinase proteins we identified are involved in this fructan utilization.

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.

Selective antibacterial and apoptosis-modulating activities of mastic.

Mastic is a resinous exudate obtained from the stem and the main leaves of Pistacia lentiscus. We have reported the antiplaque effect of mastic-containing chewing gum on the oral cavity. We hypothesize that mastic may be a multifunctional food which has some beneficial pharmaceutical properties. The aim of this study was to assess the biological activity of solid and liquid types of mastic by cytotoxicity against fibroblasts, radical-scavenging activities and inhibitory effect on cell death of oral polymorphonuclear leukocytes (OPMNs). Mastic showed selective antibacterial action against Porphyromonas gingivalis and Prevotella melaninogenica, but no anti-HIV activity. Among a total of thirteen human cell types, promyelocytic leukemia HL-60 was the most sensitive to the cytotoxicity of mastic, followed by myeloblastic leukemia (ML-1, KG-1), erythroleukemia (K-562), oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), hepatocellular carcinoma (HepG2), glioblastoma (T98G, U87MG) and normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast, most resistant). Mastic did not induce the differentiation of myelogenous leukemic cells into maturing cells with higher nitroblue tetrazolium-reducing activity, but induced apoptotic cell death, characterized by internucleosomal DNA fragmentation, caspase-3 activation and a decline in the intracellular concentration of putrescine. The cytotoxicity of mastic against leukemic cells did not diminish during its storage. On the other hand, mastic inhibited the spontaneous apoptosis of OPMNs. Mastic showed hydroxyl radical-scavenging activity. The selective antibacterial and apoptosis-modulating activity of mastic suggests its possible beneficial effects on oral health.

Anti-HIV and vitamin C-synergized radical scavenging activity of cacao husk lignin fractions.

Cacao husk lignin fractions, prepared by acid precipitation and 50% ethanol precipitation showed unexpectedly higher anti-human immunodeficiency virus (HIV) activity, as compared with the corresponding fractions from the cacao mass, amounting to the level comparable with that of popular anti-HIV compounds. The cacao husk lignin fractions also showed anti-influenza virus activity, but did not show antibacterial activity. The cacao husk lignin fractions synergistically enhanced the superoxide anion and hydroxyl radical scavenging activity of vitamin C. The cacao husk lignin fractions stimulated nitric oxide generation by mouse macrophage-like cells, to a level higher than that attained by lipopolysaccharide (LPS). The present study suggests the functionality of cacao husk lignin fractions as complementary alternative medicine.

Homogeneous enzymatic assay for L-cysteine with betaC-S lyase.

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of betaC-S lyase from Streptococcus anginosus, which catalyzes the alpha,beta-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.

High production of methyl mercaptan by L-methionine-alpha-deamino-gamma-mercaptomethane lyase from Treponema denticola.

Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.