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1.
Artigo em Inglês | MEDLINE | ID: mdl-35457601

RESUMO

BACKGROUND: Periodontal pathogens are related to the incidence of systemic diseases. This study aimed to examine whether periodontal pathogen burden is associated with the risk of fever onset in older adults. METHODS: Older adults in nursing homes, aged ≥65 years, were enrolled. The study was set in Kitakyushu, Japan. The body temperatures of participants were ≥37.2 °C and were recorded for eight months. As periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia were qualified by a real-time polymerase chain reaction at the baseline. For statistical analysis, the number of bacterial counts was logarithmically conversed to 10 as a base. RESULTS: Data from 56 participants with a median age of 88 (62-98) years were available for analysis. The logarithmic-conversed bacterial counts of T. forsythia, but not P. gingivalis or T. denticola, were associated with the onset of fever in older residents. The Kaplan-Meier method revealed that the group with <104 of T. forsythia had significantly less cumulative fever incidence than the group with ≥104 of T. forsythia. The group with ≥104 of T. forsythia was associated with an increased risk of fever onset (hazard ratio, 3.7; 98% confidence interval, 1.3-10.2; p = 0.012), which was adjusted for possible confounders. CONCLUSIONS: Bacterial burden of T. forsythia in the oral cavity was associated with the risk of the onset of fever in older nursing homes residents.


Assuntos
Tannerella forsythia , Treponema denticola , Idoso , Idoso de 80 Anos ou mais , Humanos , Casas de Saúde , Porphyromonas gingivalis , Estudos Prospectivos
2.
Pathog Dis ; 80(1)2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35404415

RESUMO

Tannerella forsythia is strongly implicated in the development of periodontitis, an inflammatory disease that destroys the bone and soft tissues supporting the tooth.  To date, the knowledge of the virulence attributes of T. forsythia species has mainly come from studies with a laboratory adapted strain (ATCC 43037). In this study, we focused on two T. forsythia clinical isolates, UB4 and UB20, in relation to their ability to activate macrophages. We found that these clinical isolates differentially induced proinflammatory cytokine expression in macrophages. Prominently, the expression of the chemokine protein IP-10 (CXCL10) was highly induced by UB20 as compared to UB4 and the laboratory strain ATCC 43037. Our study focused on the lipopolysaccharide component (LPS) of these strains and found that UB20 expressed a smooth-type LPS, unlike UB4 and ATCC 43037 each of which expressed a rough-type LPS. The LPS from UB20, via activation of TLR4, was found to be a highly potent inducer of IP-10 expression via signaling through STAT1 (signal transducer and activator of transcription-1). These data suggest that pathogenicity of T. forsythia species could be strain dependent and the LPS heterogeneity associated with the clinical strains might be responsible for their pathogenic potential and severity of periodontitis.


Assuntos
Periodontite , Tannerella forsythia , Quimiocina CXCL10/genética , Humanos , Interferon gama , Lipopolissacarídeos , Macrófagos
3.
J Bacteriol ; 204(3): e0059721, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35129368

RESUMO

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-ß-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-N-acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic ß-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic ß-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-ß-N-acetylmuramidases. IMPORTANCE Two exo-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.


Assuntos
Peptidoglicano , Tannerella forsythia , Acetilglucosamina/metabolismo , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Dissacarídeos/metabolismo , Peptidoglicano/metabolismo , Especificidade por Substrato , Tannerella forsythia/genética
4.
Front Cell Infect Microbiol ; 12: 835509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35223555

RESUMO

Tannerella forsythia and Porphyromonas gingivalis target distinct virulence factors bearing a structurally conserved C-terminal domain (CTD) to the type IX protein secretion system (T9SS). The T9SS comprises an outer membrane translocation complex which works in concert with a signal peptidase for CTD cleavage. Among prominent T9SS cargo linked to periodontal diseases are the TfsA and TfsB components of T. forsythia's cell surface (S-) layer, the bacterium's BspA surface antigen and a set of cysteine proteinases (gingipains) from P. gingivalis. To assess the overall role of the bacterial T9SS in the host response, human macrophages and human gingival fibroblasts were stimulated with T. forsythia and P. gingivalis wild-type bacteria and T9SS signal peptidase-deficient mutants defective in protein secretion, respectively. The immunostimulatory potential of these bacteria was compared by analyzing the mRNA expression levels of the pro-inflammatory mediators IL-6, IL-8, MCP-1 and TNF-α by qPCR and by measuring the production of the corresponding proteins by ELISA. Shot-gun proteomics analysis of T. forsythia and P. gingivalis outer membrane preparations confirmed that several CTD-bearing virulence factors which interact with the human immune system were depleted from the signal peptidase mutants, supportive of effective T9SS shut-down. Three and, more profoundly, 16 hours post stimulation, the T. forsythia T9SS mutant induced significantly less production of cytokines and the chemokine in human cells compared to the corresponding parent strain, while the opposite was observed for the P. gingivalis T9SS mutant. Our data indicate that T9SS shut-down translates into an altered inflammatory response in periodontal pathogens. Thus, the T9SS as a potential novel target for periodontal therapy needs further evaluation.


Assuntos
Porphyromonas gingivalis , Tannerella forsythia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Humanos , Imunidade , Tannerella forsythia/genética , Tannerella forsythia/metabolismo
5.
Lab Chip ; 22(4): 733-737, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35037006

RESUMO

The concept of time to place conversion makes using a continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes; however, it also brings about low throughput amplicons. Although multiplex PCR can simultaneously amplify more than one target gene in the chip, it may easily induce false positives because of cross-reactions. To circumvent this problem, we herein fabricated a microfluidic system based on a CF-PCR array microfluidic chip. By dividing the chip into three parts, we successfully amplified target genes of Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f) and Treponema denticola (T.d). The results demonstrated that the minimum amplification time required for P.g, T.d and T.f was 2'07'', 2'51'' and 5'32'', respectively. The target genes of P.g, T.d and T.f can be simultaneously amplified in less than 8'05''. Such a work may provide a clue to the development of a high throughput CF-PCR microfluidic system, which is crucial for point of care testing for simultaneous detection of various pathogens.


Assuntos
Microfluídica , Treponema denticola , Reação em Cadeia da Polimerase , Porphyromonas gingivalis/genética , Tannerella forsythia/genética , Treponema denticola/genética
6.
J Dent Res ; 101(4): 374-383, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34889148

RESUMO

Porphyromonas, Tannerella, and Prevotella species found in severe periodontitis use the Type IX Secretion System (T9SS) to load their outer membrane surface with an array of virulence factors. These virulence factors are then released on outer membrane vesicles (OMVs), which penetrate the host to dysregulate the immune response to establish a positive feedback loop of chronic, inflammatory destruction of the tooth's supporting tissues. In this review, we present the latest information on the molecular architecture of the T9SS and provide mechanistic insight into its role in secretion and attachment of cargo proteins to produce a virulence coat on cells and OMVs. The recent molecular structures of the T9SS motor comprising PorL and PorM as well as the secretion pore Sov, together with advances in the overall interactome, have provided insight into the possible mechanisms of secretion. We propose the presence of PorL/M motors arranged in a circle at the inner membrane with bent periplasmic rotors interacting with the PorN protein. At the outer membrane, we envisage a slide carousel model where the PorN protein is driven around a circular track composed of PorK. Cargo proteins are transported by PorN to PorW and the Sov translocon just as slides are rotated to the projection window. Secreted proteins are proposed to then be shuttled along highways consisting of the PorV shuttle protein to an array of attachment complexes distributed around the cell. The cell surface attachment of cargo is a hallmark of the T9SS, and in Porphyromonas gingivalis and Tannerella forsythia, this attachment is achieved via covalent bonding to a linking sugar synthesized by the Wbp/Vim pathway. The cell-surface attached cargo are enriched on OMVs, which are then released from the cell.


Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis , Tannerella forsythia , Fatores de Virulência
7.
Int J Mol Sci ; 22(23)2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34884826

RESUMO

IgA nephropathy (IgAN) has been considered to have a relationship with infection in the tonsil, because IgAN patients often manifest macro hematuria just after tonsillitis. In terms of oral-area infection, the red complex of periodontal bacteria (Porphyromonas gingivalis (P. gingivalis), Treponema denticol (T. denticola) and Tannerella forsythia (T. forsythia)) is important, but the relationship between these bacteria and IgAN remains unknown. In this study, the prevalence of the red complex of periodontal bacteria in tonsil was compared between IgAN and tonsillitis patients. The pathogenicity of IgAN induced by P. gingivalis was confirmed by the mice model treated with this bacterium. The prevalence of P. gingivalis and T. forsythia in IgAN patients was significantly higher than that in tonsillitis patients (p < 0.001 and p < 0.05, respectively). A total of 92% of tonsillitis patients were free from red complex bacteria, while only 48% of IgAN patients had any of these bacteria. Nasal administration of P. gingivalis in mice caused mesangial proliferation (p < 0.05 at days 28a nd 42; p < 0.01 at days 14 and 56) and IgA deposition (p < 0.001 at day 42 and 56 after administration). Scanning-electron-microscopic observation revealed that a high-density Electron-Dense Deposit was widely distributed in the mesangial region in the mice kidneys treated with P. gingivalis. These findings suggest that P. gingivalis is involved in the pathogenesis of IgAN.


Assuntos
Glomerulonefrite por IGA/patologia , Imunoglobulina A/metabolismo , Porphyromonas gingivalis/patogenicidade , Adulto , Animais , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Modelos Animais de Doenças , Feminino , Glomerulonefrite por IGA/microbiologia , Humanos , Rim/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/isolamento & purificação , Tannerella forsythia/genética , Tannerella forsythia/isolamento & purificação , Tannerella forsythia/patogenicidade , Tonsilite/microbiologia , Tonsilite/patologia , Adulto Jovem
8.
J Infect Dev Ctries ; 15(11): 1685-1693, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34898497

RESUMO

INTRODUCTION: Development of bacterial resistance and antimicrobial side-effect has shifted the focus of research toward Ethnopharmacology. A biologically active compound derived from the plants may increase the effectiveness of antibiotic when used in combination. The present study aims to determine the synergistic antibacterial effect of ethanolic extracts of Punica granatum (pericarp), Commiphora molmol, Azadirachta indica (bark) in combination with amoxicillin, metronidazole, tetracycline, and azithromycin on periodontopathic bacteria: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans. METHODOLOGY: Periodontopathic bacterial strains were isolated from the plaque sample that was collected from periodontitis patients and grown under favorable conditions. Susceptibility of bacteria to the antibiotics and extracts was determined by disc diffusion method by measuring the diameter of the inhibition zones. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts were evaluated against each bacterium. Synergistic effect of plant extract in combination with antibiotics was tested against each bacterium by measuring the diameter of zone of inhibition (ZOI). RESULTS: Findings revealed that all plant extracts exhibited an inhibitory effects on the proliferation and growth of periodontopathic bacteria. The maximum antibacterial effect was exhibited by C. molmol on P. gingivalis (ZOI = 20 ± 0.55 mm, MIC = 0.53 ± 0.24 mg/mL and MBC = 5.21 ± 1.81 mg/mL) (p < 0.05), meanwhile, no antibacterial activity was exhibited by P. granatum on T. forsythia. Synergistic antibacterial effect was recorded when plant extracts were used in combination with antibiotics. The best synergism was exhibited by P. granatum with amoxicillin against A. actinomycetemcomitans (24 ± 1.00 mm) (p < 0.05). CONCLUSIONS: The synergistic test showed significant antibacterial activity when plant extracts were combined with antibiotics against all the experimented bacteria.


Assuntos
Antibacterianos/farmacologia , Periodontite/microbiologia , Extratos Vegetais/farmacologia , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Antibacterianos/uso terapêutico , Quimioterapia Combinada , Humanos , Testes de Sensibilidade Microbiana , Periodontite/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Porphyromonas gingivalis/efeitos dos fármacos , Tannerella forsythia/efeitos dos fármacos
9.
Biomolecules ; 11(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34944439

RESUMO

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.


Assuntos
Bacteroides/crescimento & desenvolvimento , Fucosiltransferases/química , Fucosiltransferases/genética , Polissacarídeos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Bacteroides fragilis/enzimologia , Bacteroides fragilis/crescimento & desenvolvimento , Configuração de Carboidratos , Evolução Molecular , Fucosiltransferases/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosilação , Modelos Moleculares , Pedobacter/enzimologia , Pedobacter/crescimento & desenvolvimento , Polissacarídeos/metabolismo , Tannerella forsythia/enzimologia , Tannerella forsythia/crescimento & desenvolvimento
10.
mSphere ; 6(5): e0064921, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34523981

RESUMO

Tannerella forsythia is a Gram-negative oral pathogen known to possess an O-glycosylation system responsible for targeting multiple proteins associated with virulence at the three-residue motif (D)(S/T)(A/I/L/V/M/T). Multiple proteins have been identified to be decorated with a decasaccharide glycan composed of a poorly defined core plus a partially characterized species-specific section. To date, glycosylation studies have focused mainly on the two S-layer glycoproteins, TfsA and TfsB, so the true extent of glycosylation within this species has not been fully explored. In the present study, we characterize the glycoproteome of T. forsythia by employing FAIMS-based glycopeptide enrichment of a cell membrane fraction. We demonstrate that at least 13 glycans are utilized within the T. forsythia glycoproteome, varying with respect to the presence of the three terminal sugars and the presence of fucose and digitoxose residues at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides, we utilized trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. Reducing the chemical complexity of glycopeptides dramatically improved the number of glycopeptides identified and our ability to localize glycosylation sites by ETD fragmentation, leading to the identification of 312 putative glycosylation sites in 145 glycoproteins. Glycosylation site analysis revealed that glycosylation occurs on a much broader motif than initially reported, with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The prevalence of this broader glycosylation motif in the genome suggests the existence of hundreds of potential O-glycoproteins in this organism. IMPORTANCE Tannerella forsythia is an oral pathogen associated with severe forms of periodontal disease characterized by destruction of the tooth's supporting tissues, including the bone. The bacterium releases a variety of proteins associated with virulence on the surface of outer membrane vesicles. There is evidence that these proteins are modified by glycosylation, and this modification is essential for virulence in producing disease. We have utilized novel techniques coupled with mass spectrometry to identify over 13 glycans and 312 putative glycosylation sites in 145 glycoproteins within T. forsythia. Glycosylation site analysis revealed that this modification occurs on a much broader motif than initially reported such that there is a high prevalence of potential glycoproteins in this organism that may help to explain its role in periodontal disease.


Assuntos
Proteínas de Bactérias/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoma/metabolismo , Tannerella forsythia/metabolismo , Proteínas de Bactérias/química , Glicosilação , Espectrometria de Massas , Glicoproteínas de Membrana/química , Mesilatos/farmacologia , Transporte Proteico , Tannerella forsythia/efeitos dos fármacos , Tannerella forsythia/genética , Tannerella forsythia/patogenicidade , Virulência
11.
Biomed Res Int ; 2021: 9986375, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222492

RESUMO

Increasing attention has been paid to the possible link between periodontal disease and atherosclerosis over the past decade. The aim of this study is to investigate the presence of five periopathogens: Porphyromonas gingivalis (P.g.), Aggregatibacter actinomycetemcomitans (A.a.), Tannerella forsythia (T.f.), Treponema denticola (T.d.), and Prevotella intermedia (P.i.) in atheromatous plaques obtained from the carotid and coronary arteries in patients who underwent coronary artery bypass graft surgery and carotid endarterectomy. Group I (carotid arteries) consisted of 30 patients (mean age: 54.5 ± 14.8), and group II (coronary arteries) consisted of 28 patients (mean age: 63 ± 12.1). Clinical periodontal examinations consisted of plaque index, gingival index, sulcus bleeding index, and periodontal probing depth and were performed on the day of vascular surgery. The presence of periopathogens in periodontal pockets and atherosclerotic vessels was detected using polymerase chain reaction. In both subgingival plaque and atherosclerotic plaque of carotid arteries, P.g., A.a., T.f., T.d., and P.i. were detected in 26.7%, 6.7%, 66.7%, 10.0%, and 20.0%, respectively, while for coronary arteries, P.g. was detected in 39.3%, A.a. in 25%, T.f. in 46.4%, T.d. in 7.1%, and P.i. in 35.7%. The presence of five periopathogens in carotid and coronary atherosclerotic vessels showed correlation in regard to the degree of periodontal inflammation. The present study suggests the relationship between periodontal pathogenic bacteria and atherogenesis. Further studies are necessary in relation to the prevention or treatment of periodontal disease that would result in reduced mortality and morbidity associated with atherosclerosis.


Assuntos
Artérias Carótidas/microbiologia , Vasos Coronários/microbiologia , Placa Aterosclerótica/microbiologia , Adulto , Idoso , Aggregatibacter actinomycetemcomitans , Aterosclerose , Feminino , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Doenças Periodontais/imunologia , Reação em Cadeia da Polimerase , Porphyromonas gingivalis , Prevotella intermedia , RNA Ribossômico 16S/metabolismo , Fatores de Risco , Tannerella forsythia , Treponema denticola
12.
J Enzyme Inhib Med Chem ; 36(1): 1267-1281, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34210221

RESUMO

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.


Assuntos
Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Inibidores de Proteases/farmacologia , Tannerella forsythia/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/química , Tannerella forsythia/isolamento & purificação , Temperatura
13.
Microb Physiol ; 31(2): 123-134, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34107471

RESUMO

Tannerella forsythia is an anaerobic, fusiform Gram-negative oral pathogen strongly associated with periodontitis, a multibacterial inflammatory disease that leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. To survive in the oral habitat, T. forsythia depends on cohabiting bacteria for the provision of nutrients. For axenic growth under laboratory conditions, it specifically relies on the external supply of N-acetylmuramic acid (MurNAc), which is an essential constituent of the peptidoglycan (PGN) of bacterial cell walls. T. forsythia comprises a typical Gram-negative PGN; however, as evidenced by genome sequence analysis, the organism lacks common enzymes required for the de novo synthesis of precursors of PGN, which rationalizes its MurNAc auxotrophy. Only recently insights were obtained into how T. forsythia gains access to MurNAc in its oral habitat, enabling synthesis of the own PGN cell wall. This report summarizes T. forsythia's strategies to survive in the oral habitat by means of PGN salvage pathways, including recovery of exogenous MurNAc and PGN-derived fragments but also polymeric PGN, which are all derived from cohabiting bacteria either via cell wall turnover or decay of cells. Salvage of polymeric PGN presumably requires the removal of peptides from PGN by an unknown amidase, concomitantly with the translocation of the polymer across the outer membrane. Two recently identified exo-lytic N-acetylmuramidases (Tf_NamZ1 and Tf_NamZ2) specifically cleave the peptide-free, exogenous (nutrition source) PGN in the periplasm and release the MurNAc and disaccharide substrates for the transporters Tf_MurT and Tf_AmpG, respectively, whereas the peptide-containing, endogenous (the self-cell wall) PGN stays unattached. This review also outlines how T. forsythia synthesises the PGN precursors UDP-MurNAc and UDP-N-acetylglucosamine (UDP-GlcNAc), involving homologs of the Pseudomonas sp. recycling enzymes AmgK/MurU and a monofunctional uridylyl transferase (named Tf_GlmU*), respectively.


Assuntos
Microbiota , Peptidoglicano , Parede Celular , Tannerella , Tannerella forsythia
14.
Lab Chip ; 21(16): 3159-3164, 2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34190300

RESUMO

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.


Assuntos
Microfluídica , Treponema denticola , Adulto , Humanos , Reação em Cadeia da Polimerase , Porphyromonas gingivalis/genética , Tannerella forsythia , Treponema denticola/genética
15.
Glycobiology ; 31(9): 1176-1191, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-33909048

RESUMO

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.


Assuntos
Acetilesterase , Ácido N-Acetilneuramínico , Acetilação , Acetilesterase/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Tannerella forsythia
16.
J Biol Chem ; 296: 100263, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33837744

RESUMO

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.


Assuntos
Aminoaciltransferases/química , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Prevotella intermedia/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Aminoaciltransferases/ultraestrutura , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Periodontite/tratamento farmacológico , Periodontite/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , Estrutura Terciária de Proteína/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Tannerella forsythia/enzimologia , Tannerella forsythia/patogenicidade
17.
J Periodontol ; 92(8): e94-e102, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33543507

RESUMO

BACKGROUND: Translocation of periodontal pathogens into the respiratory tract could either cause pneumonia or disrupt local defense mechanisms, predisposing the host to infection by respiratory pathogens. The objective of this pilot study was to evaluate the levels of periodontopathogenic bacteria in subglottic samples of intubated and mechanically ventilated patients and the impact of oral decontamination with chlorhexidine (CHX) on subglottic levels of these microorganisms. METHODS: Patients scheduled to undergo elective surgical procedures requiring endotracheal intubation and mechanical ventilation for at least 3 hours were included. Following full-mouth periodontal examination, patients were randomly assigned to groups that rinsed preoperatively with 0.12% CHX or 0.9% saline (control). After 3 hours of orotracheal intubation, subglottic contents were collected. Quantification of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Tannerella forsythia (T. forsythia) in subglottic samples was determined using quantitative real-time polymerase chain reaction. Data were analyzed by Fisher Exact Probability, unpaired Student's t and Mann-Whitney tests. RESULTS: Of the 69 patients included, 43 completed study participation. There were no differences between control and CHX groups in subglottic detection rates and abundance levels of P. gingivalis (P = 0.59), T. forsythia (P = 0.83) and A. actinomycetemcomitans (P = 0.07). Moreover, our data indicate that periodontal health has no impact on subglottic levels of P. gingivalis, T. forsythia, and A. actinomycetemcomitans. CONCLUSIONS: Periodontal pathogens were detected in subglottic samples of intubated and mechanically ventilated patients. Moreover, a single CHX rinse prior to endotracheal intubation may have no effect on subglottic contamination by P. gingivalis, T. forsythia, and A. actinomycetemcomitans.


Assuntos
Anestesia Geral , Clorexidina , Procedimentos Cirúrgicos Eletivos , Intubação Intratraqueal , Laringe/microbiologia , Antissépticos Bucais , Aggregatibacter actinomycetemcomitans , Humanos , Projetos Piloto , Porphyromonas gingivalis , Tannerella forsythia
18.
Arch Oral Biol ; 124: 105062, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33524879

RESUMO

OBJECTIVES: The study aimed to examine the diverse bioactivity of lipooligosaccharide extracted from T. denticola cultured in the presence of hemin and quorum-sensing inhibitor. DESIGN: T. denticola was cultured in the presence or absence hemin or 2(5 H)-furanone, and lipooligosaccharide from T. denticola cultured in various conditions was extracted using an extraction kit. To investigate bioactivity of the lipooligosaccharide, human gingival fibroblasts (HGFs) were treated with the extracted lipooligosaccharide in the presence or absence of Tannerella forsythia lipopolysaccharide. The induction of cytokine expressions was investigated by real-time RT-PCR and ELISA, and the signaling pathway was examined by immunoblotting. To investigate antagonistic mechanisms of the lipooligosaccharide, HGFs were cotreated with fluorescence-labeled T. forsythia lipopolysaccharide and the extracted lipooligosaccharide. Binding of T. forsythia lipopolysaccharide to the cell was analyzed by a flow cytometer. RESULTS: Lipooligosaccharide induced a low level of cytokine expression at high concentration of hemin or 2(5 H)-furanone. Lipooligosaccharide extracted from T. denticola cultured in higher concentration of hemin and 2(5 H)-furanone had a greater inhibitory effect on induction of cytokine expression by T. forsythia lipopolysaccharide. Further, lipooligosaccharide inhibited the activation of NF-κB and mitogen-activated protein kinase signaling pathways by T. forsythia lipopolysaccharide. Lipooligosaccharide inhibited the binding of T. forsythia lipopolysaccharide to HGFs in the presence of CD14 and LBP. CONCLUSIONS: The characteristics of T. denticola lipooligosaccharide may be altered by bacterial communication and host factors.


Assuntos
Lipopolissacarídeos , Treponema denticola , Hemina/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Tannerella forsythia
19.
Cancer ; 127(4): 512-519, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33156979

RESUMO

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.


Assuntos
Placa Dentária/microbiologia , Neoplasias Esofágicas/microbiologia , Boca/microbiologia , Saliva/microbiologia , Adulto , Idoso , Aggregatibacter actinomycetemcomitans , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/etiologia , Feminino , Fusobacterium nucleatum/isolamento & purificação , Fusobacterium nucleatum/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Porphyromonas gingivalis/isolamento & purificação , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/isolamento & purificação , Prevotella intermedia/patogenicidade , Fatores de Risco , Streptococcus anginosus/isolamento & purificação , Streptococcus anginosus/patogenicidade , Tannerella forsythia/isolamento & purificação , Tannerella forsythia/patogenicidade , Treponema denticola/isolamento & purificação , Treponema denticola/patogenicidade
20.
Odontology ; 109(2): 484-493, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33145632

RESUMO

The effect of periodontal treatment on clinical, microbiological and serological parameters of patients with rheumatoid arthritis (RA) are scarce and controversial. The aim of this study was to investigate the influence of non-surgical periodontal treatment on clinical periodontal status, subgingival bacterial levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and RA activity through a controlled clinical trial on individuals with RA and periodontitis (PE). From a convenience sample, 107 individuals were considered eligible and consecutively allocated in four groups: (1) individuals without PE and RA (- PE-RA, n = 30); (2) individuals without PE and with RA (- PE + RA, n = 23); (3) individuals with PE and RA (+ PE + RA, n = 24); and (4) individuals with PE and without RA (+ PE-RA, n = 30). Full-mouth periodontal clinical examinations, microbiological analysis and Disease Activity Score (DAS-28) evaluations were performed at baseline (T1) and 45 days after non-surgical periodontal treatment (T2). At T1, individuals + PE + RA showed greater severity of PE than + PE-RA individuals. At T2, significant reductions were observed in all periodontal clinical parameters in both groups (p < 0.001) with a significant reduction in DAS-28 in + PE + RA (p = 0.011). Individuals + PE-RA and + PE-RA showed significant reductions for all bacteria (p < 0.001). Additionally, P. gingivalis demonstrated an expressively significant reduction in + PE + RA (p < 0.001). Non-surgical periodontal treatment was effective on improving the clinical periodontal condition, improving the RA clinical status and reducing the presence of periodontal pathogens. Brazilian Registry of Clinical Trials (ReBEC) protocol #RBR-8g2bc8 ( https://www.ensaiosclinicos.gov.br/rg/RBR-8g2bc8/ ).


Assuntos
Artrite Reumatoide , Treponema denticola , Aggregatibacter actinomycetemcomitans , Artrite Reumatoide/terapia , Brasil , Humanos , Porphyromonas gingivalis , Tannerella forsythia
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