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1.
Int J Oral Sci ; 13(1): 31, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593756

RESUMO

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.


Assuntos
Colite Ulcerativa , Porphyromonas gingivalis , Desiminases de Arginina em Proteínas , Animais , Colite Ulcerativa/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/patogenicidade , Fatores de Virulência
2.
PLoS Pathog ; 17(5): e1009598, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015051

RESUMO

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteasome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production.


Assuntos
Infecções por Bacteroidaceae/microbiologia , Doenças da Gengiva/microbiologia , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Animais , Ciclo Celular , Movimento Celular , Proliferação de Células , Células Epiteliais/microbiologia , Transição Epitelial-Mesenquimal , Gengiva/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Porphyromonas gingivalis/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima
3.
Biol Chem ; 402(7): 759-768, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33823093

RESUMO

Porphyromonas gingivalis is a bacterial species known to be involved in the pathogenesis of chronic periodontitis, that more recently has been as well associated with Alzheimer's disease. P. gingivalis expresses a glutaminyl cyclase (PgQC) whose human ortholog is known to participate in the beta amyloid peptide metabolism. We have elucidated the crystal structure of PgQC at 1.95 Å resolution in unbound and in inhibitor-complexed forms. The structural characterization of PgQC confirmed that PgQC displays a mammalian fold rather than a bacterial fold. Our biochemical characterization indicates that PgQC uses a mammalian-like catalytic mechanism enabled by the residues Asp149, Glu182, Asp183, Asp218, Asp267 and His299. In addition, we could observe that a non-conserved Trp193 may drive differences in the binding affinity of ligands which might be useful for drug development. With a screening of a small molecule library, we have identified a benzimidazole derivative rendering PgQC inhibition in the low micromolar range that might be amenable for further medicinal chemistry development.


Assuntos
Aminoaciltransferases/química , Porphyromonas gingivalis/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Benzimidazóis/química , Benzimidazóis/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares
4.
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
5.
mBio ; 12(1)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622730

RESUMO

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two ß-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the "attachment complex" composed of PorU, PorZ, and the integral OM ß-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Cisteína Endopeptidases/metabolismo , Lipopolissacarídeos/metabolismo , Peptidil Transferases/metabolismo , Porphyromonas gingivalis/genética , Sistemas de Secreção Bacterianos/genética , Peptidil Transferases/genética , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico
6.
J Bacteriol ; 203(4)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33257525

RESUMO

Many bacteria switch between a sessile and a motile mode in response to environmental and host-related signals. Porphyromonas gingivalis, an oral anaerobe implicated in the etiology of chronic periodontal disease, has long been described as a nonmotile bacterium. And yet, recent studies have shown that under certain conditions, P. gingivalis is capable of surface translocation. Considering these findings, this work aimed to increase our understanding of how P. gingivalis transitions between sessile growth and surface migration. Here, we show that the peptidylarginine deiminase secreted by P. gingivalis (PPAD), an enzyme previously shown to be upregulated during surface translocation and to constrain biofilm formation, promotes surface translocation. In the absence of PPAD, the production of outer membrane vesicles (OMVs) was drastically reduced. In turn, there was a reduction in gingipain-mediated proteolysis and a reduced zone of hydration around the site of inoculation. Transcriptome sequencing (RNA-Seq) and metabolomics analyses also showed that these changes corresponded to a shift in arginine metabolism. Overall, this report provides new evidence for the functional relevance of PPAD and proteases, as well as the importance of PPAD activity in OMV biogenesis and release. Our findings support the model that citrullination is a critical mechanism during lifestyle transition between surface-attached growth and surface translocation by modulating OMV-mediated proteolysis and arginine metabolism.IMPORTANCE Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production, and many of the proteins packaged in these vesicles are proteolytic or protein-modifying enzymes. This includes key virulence determinants, such as the gingipains and PPAD (a unique peptidylarginine deiminase). Here, we show that PPAD activity (citrullination) is involved in OMV biogenesis. The study revealed an unusual mechanism that allows this bacterium to transform its surroundings. Since OMVs are detected in circulation and in systemic tissues, our study results also support the notion that PPAD activity may be a key factor in the correlation between periodontitis and systemic diseases, further supporting the idea of PPAD as an important therapeutic target.


Assuntos
Arginina/metabolismo , Membrana Externa Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Porphyromonas gingivalis/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/genética
7.
Appl Environ Microbiol ; 87(3)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33158898

RESUMO

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and reestablish a health-associated microbiota, a high-throughput in vitro multispecies biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rRNA gene amplicon sequencing as well as measurement of dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principal-component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis to saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder a microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies.IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed targeting reversing dysbiosis and restoring host-compatible microbiota rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in a dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large-scale evaluation of strategies that can potentially modulate microbial ecology.


Assuntos
Disbiose/microbiologia , Gengiva/microbiologia , Porphyromonas gingivalis/fisiologia , Saliva/microbiologia , Biofilmes , Ácido Butírico/metabolismo , Dipeptidil Peptidase 4/metabolismo , Humanos , Microbiota/genética , Microbiota/fisiologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética , RNA Ribossômico 16S/genética
8.
Front Immunol ; 11: 1551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32793214

RESUMO

Background: Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation. Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots. Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting. Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Cisteína Endopeptidases Gingipaínas/química , Cisteína Endopeptidases Gingipaínas/farmacologia , Fenômenos Mecânicos , Porphyromonas gingivalis/enzimologia , Adulto , Feminino , Fibrina/química , Fibrinogênio/química , Fibrinogênio/ultraestrutura , Humanos , Lipopolissacarídeos/efeitos adversos , Masculino , Microscopia de Força Atômica , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas Recombinantes , Reologia , Análise Espectral Raman , Trombose/tratamento farmacológico , Adulto Jovem
9.
Mol Oral Microbiol ; 35(5): 222-230, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32794617

RESUMO

Uric acid is a potential metabolite that serves as a danger-associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP-1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase-1 activation, cell death, and the expression of proinflammatory cytokines, including IL-1α, IL-6, and IL-8, in the human keratinocyte HOK-16B cell line. Our results suggest that gingipain-induced uric acid can mediate inflammation in periodontal tissue cells.


Assuntos
Cisteína Endopeptidases Gingipaínas/metabolismo , Porphyromonas gingivalis/enzimologia , Ácido Úrico/metabolismo , Linhagem Celular , Citocinas/metabolismo , Humanos , Inflamação , Queratinócitos , Porphyromonas gingivalis/patogenicidade , Células THP-1 , Xantina Desidrogenase/metabolismo
10.
Arthritis Res Ther ; 22(1): 114, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32410713

RESUMO

BACKGROUND: Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of auto-inflammatory diseases. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant bone loss in model systems and in patients. OBJECTIVE: We sought to gain further insight into the role of this pathobiont in rheumatoid arthritis (RA) and to identify novel therapeutic targets for auto-inflammatory diseases. METHODS: We profiled the antibody response to RgPA-specific domains in patient sera. We also tested the potential protective effects of RgpA domains in an experimental arthritis model. RESULTS: Pre-immunization of rats with purified recombinant RgpA domains alleviated arthritis in the joints of the rodents and reduced bone erosion. Using a functional genomics approach at both the mRNA and protein levels, we report that the pre-immunizations reduced arthritis severity by impacting a matrix metalloprotease characteristic of articular injury, a chemokine known to be involved in recruiting inflammatory cells, and three inflammatory cytokines. Finally, we identified an amino acid motif in the RgpA catalytic domain of P. gingivalis that shares sequence homology with type II collagen. CONCLUSION: We conclude that pre-immunization against gingipain domains can reduce the severity of experimentally induced arthritis. We suggest that targeting gingipain domains by pre-immunization, or, possibly, by small-molecule inhibitors, could reduce the potential of P. gingivalis to translocate to remote tissues and instigate and/or exacerbate pathology in RA, but also in other chronic inflammatory diseases.


Assuntos
Artrite/terapia , Cisteína Endopeptidases Gingipaínas/antagonistas & inibidores , Porphyromonas gingivalis/enzimologia , Proteínas Recombinantes/farmacologia , Animais , Domínio Catalítico , Humanos , Ratos
11.
Int J Mol Sci ; 21(7)2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32260245

RESUMO

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Cininogênios/metabolismo , Plasminogênio/metabolismo , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/farmacologia , Proteínas de Bactérias/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Cromatografia Líquida , Citrulinação , Humanos , Imunidade Inata , Cininogênios/química , Ligação Proteica , Espectrometria de Massas em Tandem
12.
Microbiol Mol Biol Rev ; 84(1)2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31896547

RESUMO

Porphyromonas gingivalis is an oral pathogen involved in the widespread disease periodontitis. In recent years, however, this bacterium has been implicated in the etiology of another common disorder, the autoimmune disease rheumatoid arthritis. Periodontitis and rheumatoid arthritis were known to correlate for decades, but only recently a possible molecular connection underlying this association has been unveiled. P. gingivalis possesses an enzyme that citrullinates certain host proteins and, potentially, elicits autoimmune antibodies against such citrullinated proteins. These autoantibodies are highly specific for rheumatoid arthritis and have been purported both as a symptom and a potential cause of the disease. The citrullinating enzyme and other major virulence factors of P. gingivalis, including some that were implicated in the etiology of rheumatoid arthritis, are targeted to the host tissue as secreted or outer-membrane-bound proteins. These targeting events play pivotal roles in the interactions between the pathogen and its human host. Accordingly, the overall protein sorting and secretion events in P. gingivalis are of prime relevance for understanding its full disease-causing potential and for developing preventive and therapeutic approaches. The aim of this review is therefore to offer a comprehensive overview of the subcellular and extracellular localization of all proteins in three reference strains and four clinical isolates of P. gingivalis, as well as the mechanisms employed to reach these destinations.


Assuntos
Artrite Reumatoide/microbiologia , Interações Hospedeiro-Patógeno , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Transporte Proteico , Artrite Reumatoide/etiologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Citrulinação/imunologia , Humanos , Boca/microbiologia , Periodontite/imunologia , Porphyromonas gingivalis/patogenicidade , Fatores de Virulência
13.
Anaerobe ; 61: 102140, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31838319

RESUMO

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis.


Assuntos
Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/microbiologia , Cisteína Endopeptidases Gingipaínas/metabolismo , Imunoglobulina G/imunologia , Fragmentos de Peptídeos/metabolismo , Periodontite/etiologia , Porphyromonas gingivalis/enzimologia , Infecções por Bacteroidaceae/imunologia , Bases de Dados de Proteínas , Suscetibilidade a Doenças , Epitopos/imunologia , Feminino , Cisteína Endopeptidases Gingipaínas/química , Cisteína Endopeptidases Gingipaínas/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Porphyromonas gingivalis/imunologia , Transporte Proteico , Relação Estrutura-Atividade
14.
Adv Exp Med Biol ; 1197: 79-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31732936

RESUMO

Porphyromonas gingivalis is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which P. gingivalis could abrogate the host-microbe symbiotic relationship leading to oral dysbiosis remain unclear. We have recently demonstrated that P. gingivalis specifically increased the antimicrobial properties of oral epithelial cells, through a strong induction of the expression of PLA2-IIA in a mechanism that involves activation of the Notch-1 receptor. Moreover, gingival expression of PLA2-IIA was significantly increased during initiation and progression of periodontal disease in non-human primates and interestingly, those PLA2-IIA expression changes were concurrent with oral dysbiosis. In this chapter, we present an innovative hypothesis of a potential mechanism involved in P. gingivalis-induced oral dysbiosis and inflammation based on our previous observations and a robust body of literature that supports the antimicrobial and proinflammatory properties of PLA2-IIA as well as its role in other chronic inflammatory diseases.


Assuntos
Disbiose , Doenças Periodontais , Porphyromonas gingivalis , Animais , Disbiose/microbiologia , Doenças Periodontais/enzimologia , Doenças Periodontais/microbiologia , Fosfolipases/genética , Poliésteres , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética
15.
Microbiology (Reading) ; 165(11): 1181-1197, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517596

RESUMO

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Neuraminidase/metabolismo , Porphyromonas gingivalis/enzimologia , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interações Microbianas , Mucinas/metabolismo , Mutação , Neuraminidase/genética , Polissacarídeos/metabolismo , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Tannerella forsythia/enzimologia , Fatores de Virulência/genética , Zanamivir/farmacologia
16.
Sci Rep ; 9(1): 13587, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537874

RESUMO

Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.


Assuntos
Benzoatos/farmacologia , Ácido Cítrico/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Porphyromonas gingivalis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzoatos/química , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Fosfatos de Inositol , Modelos Moleculares , Conformação Proteica
17.
Nutrients ; 11(9)2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527555

RESUMO

BACKGROUND: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). METHODS: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0-24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. RESULTS: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. CONCLUSIONS: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.


Assuntos
Antibacterianos/farmacologia , Infecções por Bacteroidaceae/prevenção & controle , Ácido Clorogênico/farmacologia , Coffea/química , Periodontite/tratamento farmacológico , Extratos Vegetais/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/metabolismo , Infecções por Bacteroidaceae/microbiologia , Ácido Clorogênico/isolamento & purificação , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Viabilidade Microbiana/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Extratos Vegetais/isolamento & purificação , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/patogenicidade , Sementes/química , Fatores de Tempo , Fatores de Virulência/metabolismo
18.
Eur J Oral Sci ; 127(4): 287-293, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175838

RESUMO

Arginine-specific cysteine proteinases, such as Arg-gingipain B (RgpB), mediate inflammation by activating protease-activated receptors (PARs). Arg-gingipain B is produced by Porphyromonas gingivalis, and is implicated in the causation of periodontal disease. The purpose of the present study was to observe the influence of recombinant RgpB protein (rRgpB) on PAR activation by monitoring intracellular Ca2+ ion concentration ([Ca2+]i) and inositol-1,4,5-triphosphate (IP3) levels in human gingival fibroblasts (HGFs). Our findings showed that rRgpB could cause a transient increase in [Ca2+]i. This increase in [Ca2+]i was completely suppressed by vorapaxar, a PAR-1 antagonist. Recombinant Arg-gingipain B increased the concentration of IP3, reaching a maximum at 60 s after treatment; this was completely inhibited by vorapaxar. We therefore conclude that rRgpB-induced calcium signaling in HGFs is mainly caused by PAR-1 activation. This suggests that PAR-1 activation plays a significant role in chronic inflammatory periodontal disease induced by P. gingivalis RgpB.


Assuntos
Sinalização do Cálcio , Fibroblastos/metabolismo , Cisteína Endopeptidases Gingipaínas/farmacologia , Porphyromonas gingivalis/enzimologia , Receptor PAR-1/metabolismo , Proteínas de Bactérias/farmacologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Inositol 1,4,5-Trifosfato , Lactonas/farmacologia , Piridinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Proteínas Recombinantes/farmacologia
19.
Biochimie ; 166: 161-172, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31212040

RESUMO

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.


Assuntos
Precursores Enzimáticos/metabolismo , Cisteína Endopeptidases Gingipaínas/química , Cisteína Endopeptidases Gingipaínas/metabolismo , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Processamento de Proteína Pós-Traducional , Via Secretória
20.
Biochimie ; 163: 50-57, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078584

RESUMO

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.


Assuntos
Peptídeo Hidrolases/metabolismo , Porphyromonas gingivalis/enzimologia , Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Hidrólise , Cinética , Peptídeo Hidrolases/efeitos dos fármacos , Especificidade por Substrato
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