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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Int J Mol Sci ; 20(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052493

RESUMO

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer's disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/microbiologia , Citrulinação/efeitos dos fármacos , Descoberta de Drogas , Histonas/metabolismo , Humanos , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
11.
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
12.
Biosci Biotechnol Biochem ; 83(7): 1382-1384, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30912703

RESUMO

Gingipains are potent virulence cysteine proteases secreted by Porphyromonas gingivalis, a major pathogen of periodontitis. We previously reported that epimedokoreanin B inhibits the activities of gingipains. In this report, we show that epimedokoreanin B inhibits the virulence of gingipains-containing P. gingivalis culture supernatants, indicating the potential use of this prenylated flavonoid as a new agent to combat against periodontal pathogens.


Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Flavonoides/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/enzimologia , Inibidores de Cisteína Proteinase/química , Flavonoides/química , Cisteína Endopeptidases Gingipaínas , Humanos , Porphyromonas gingivalis/patogenicidade , Virulência
13.
Sci Adv ; 5(1): eaau3333, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30746447

RESUMO

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/microbiologia , Infecções por Bacteroidaceae/tratamento farmacológico , Encéfalo/microbiologia , Encéfalo/patologia , Fármacos Neuroprotetores/uso terapêutico , Porphyromonas gingivalis/enzimologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Idoso , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Cisteína Endopeptidases Gingipaínas/antagonistas & inibidores , Cisteína Endopeptidases Gingipaínas/metabolismo , Cisteína Endopeptidases Gingipaínas/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Projetos Piloto , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/genética , Estudos Prospectivos , Saliva/microbiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas tau/metabolismo
14.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670550

RESUMO

Although the periodontal pathogen Porphyromonas gingivalis must withstand high levels of nitrosative stress while in the oral cavity, the mechanisms of nitrosative stress defense are not well understood in this organism. Previously we showed that the transcriptional regulator HcpR plays a significant role in defense, and here we further defined its regulon. Our study shows that hcp (PG0893), a putative nitric oxide (NO) reductase, is the only gene significantly upregulated in response to nitrite (NO2) and that this regulation is dependent on HcpR. An isogenic mutant deficient in hcp is not able to grow with 200 µM nitrite, demonstrating that the sensitivity of the HcpR mutant is mediated through Hcp. We further define the molecular mechanisms of HcpR interaction with the hcp promoter through mutational analysis of the inverted repeat present within the promoter. Although other putative nitrosative stress protection mechanisms present on the nrfAH operon are also found in the P. gingivalis genome, we show that their gene products play no role in growth of the bacterium with nitrite. As growth of the hcp-deficient strain was also significantly diminished in the presence of a nitric oxide-producing compound, S-nitrosoglutathione (GSNO), Hcp appears to be the primary means by which P. gingivalis responds to NO2 --based stress. Finally, we show that Hcp is required for survival with host cells but that loss of Hcp has no effect on association and entry of P. gingivalis into human oral keratinocytes.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Bacteroidaceae/microbiologia , Nitritos/metabolismo , Oxirredutases/metabolismo , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Viabilidade Microbiana , Óperon , Oxirredutases/genética , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo
15.
Protein Sci ; 28(3): 478-486, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30638292

RESUMO

Citrullination is an essential post-translational modification in which the guanidinium group of protein and peptide arginines is deiminated by peptidylarginine deiminases (PADs). When deregulated, excessive citrullination leads to inflammation as in severe periodontal disease (PD) and rheumatoid arthritis (RA). Porphyromonas gingivalis is the major periodontopathogenic causative agent of PD and also an etiological agent of RA. It secretes a PAD, termed Porphyromonas PAD (PPAD), which is a virulence factor that causes aberrant citrullination. Analysis of P. gingivalis genomes of laboratory strains and clinical isolates unveiled a PPAD variant (PPAD-T2), which showed three amino-acid substitutions directly preceding catalytic Residue H236 (G231 N/E232 T/N235 D) when compared with PPAD from the reference strain (PPAD-T1). Mutation of these positions in the reference strain resulted in twofold higher cell-associated citrullinating activity. Similar to PPAD-T1, recombinant PPAD-T2 citrullinated arginines at the C-termini of general peptidic substrates but not within peptides. Catalytically, PPAD-T2 showed weaker substrate binding but higher turnover rates than PPAD-T1. In contrast, no differences were found in thermal stability. The 1.6 Å-resolution X-ray crystal structure of PPAD-T2 in complex with the general human PAD inhibitor, Cl-amidine, revealed that the inhibitor moiety is tightly bound and that mutations localize to a loop engaged in substrate/inhibitor binding. In particular, mutation G231 N caused a slight structural rearrangement, which probably originated the higher substrate turnover observed. The present data compare two natural PPAD variants and will set the pace for the design of specific inhibitors against P. gingivalis-caused PD.


Assuntos
Inibidores Enzimáticos/farmacologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Substituição de Aminoácidos , Infecções por Bacteroidaceae/tratamento farmacológico , Infecções por Bacteroidaceae/microbiologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Porphyromonas gingivalis/química , Conformação Proteica , Desiminases de Arginina em Proteínas/química , Desiminases de Arginina em Proteínas/metabolismo
16.
Arch Oral Biol ; 97: 223-230, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399509

RESUMO

OBJECTIVES: Human glutaminyl cyclases (QC and isoQC) play an important role in maintaining inflammatory conditions. Meanwhile a glutaminyl cyclase synthesized by Porphyromonas gingivalis (PgQC), a key pathogen in developing periodontitis and a potential link of periodontitis with rheumatoid arthritis (RA), was discovered. This study was aimed to determine the expression of QC, isoQC and PgQC in patients with chronic periodontitis (CP) and RA. DESIGN: Thirty volunteers were enrolled in a pilot study and divided into 3 groups (healthy, CP and RA individuals). Blood samples, biofilm and gingival crevicular fluid (GCF) were analysed for mRNA expression of QC, isoQC and P. gingivalis QC. Major bacteria being associated with periodontal disease were quantified in subgingival biofilm and protein levels for monocyte chemoattractant protein (MCP)-1, MCP-3 and interleukin (IL)-1ß) were determined in the GCF. Expression of PgQC on the mRNA and protein levels was assessed in two P. gingivalis strains. RESULTS: PgQC is expressed in P. gingivalis strains and the protein seems to be located mainly in peri-plasmatic space. mRNA expression of QC was significantly increased in the peripheral blood from RA patients vs. healthy subjects and CP patients (p = 0.013 and p = 0.003, respectively). In GCF of RA patients, QC mRNA was detected more frequently than in healthy controls (p = 0.043). In these samples IL-1ß levels were also elevated compared to GCF from periodontally healthy individuals (p = 0.003). PgQC was detected in eight out of the 13 P. gingivalis positive biofilm samples. CONCLUSION: Activity of QC may play a supportive role in maintaining chronic periodontal inflammation and destruction in RA. PgQC is expressed in vivo but further research is needed to evaluate biological importance of this enzyme and if it constitutes a potential target in periodontal antimicrobial therapy.


Assuntos
Aminoaciltransferases/metabolismo , Artrite Reumatoide/metabolismo , Periodontite Crônica/metabolismo , Adulto , Idoso , Western Blotting , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Líquido do Sulco Gengival/química , Humanos , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Periodonto/metabolismo , Projetos Piloto , Porphyromonas gingivalis/enzimologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
17.
NPJ Biofilms Microbiomes ; 5(1): 7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029738

RESUMO

Porphyromonas gingivalis is the only known human-associated prokaryote that produces a peptidylarginine deiminase (PPAD), a protein-modifying enzyme that is secreted along with a number of virulence factors via a type IX secretion system (T9SS). While the function of PPAD in P. gingivalis physiology is not clear, human peptidylarginine deiminases are known to convert positively charged arginine residues within proteins to neutral citrulline and, thereby, impact protein conformation and function. Here, we report that the lack of citrullination in a PPAD deletion mutant (Δ8820) enhances biofilm formation. More Δ8820 cells attached to the surface than the parent strain during the early stages of biofilm development and, ultimately, mature Δ8820 biofilms were comprised of significantly more cell-cell aggregates and extracellular matrix. Imaging by electron microscopy discovered that Δ8820 biofilm cells secrete copious amounts of protein aggregates. Furthermore, gingipain-derived adhesin proteins, which are also secreted by the T9SS were predicted by mass spectrometry to be citrullinated and citrullination of these targets by wild-type strain 381 in vitro was confirmed. Lastly, Δ8820 biofilms contained more gingipain-derived adhesin proteins and more gingipain activity than 381 biofilms. Overall, our findings support the model that citrullination of T9SS cargo proteins known to play a key role in colonization, such as gingipain-derived adhesin proteins, is an underlying mechanism that modulates P. gingivalis biofilm development.


Assuntos
Biofilmes/crescimento & desenvolvimento , Citrulinação , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/fisiologia , Adesinas Bacterianas/metabolismo , Arginina/metabolismo , Citrulina/metabolismo , Deleção de Genes , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo
18.
Autoimmunity ; 51(6): 304-309, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30417696

RESUMO

Anti-citrullinated protein autoantibodies (ACPA) precede the onset of clinical and subclinical rheumatoid arthritis (RA). ACPA are frequently generated in further chronic inflammatory diseases, e.g. chronic obstructive pulmonary disease, lupus, periodontitis (PD), characterized by citrullination and mucosal as well as systemic autoimmunity against citrullinated proteins. PD is of particular interest, as it exhibits two sources of citrullination, namely peptidylarginine deiminase 4 (PAD4) of periodontal neutrophils and neutrophil extracellular traps (NETs) as well as the PAD of Porphyromonas gingivalis (PPAD). Whereas the PAD4-citrullinated host peptides and/or proteins occur physiologically, PPAD-citrullinated ones appear under pathological conditions as neo-antigens. Frequently, the oral pathogens P. gingivalis and A. actinomycetemcomitans directly and indirectly participate in synovitis in RA, providing topical citrullination: P. gingivalis via PPAD and A. actinomycetemcomitans via leukotoxin A-mediated ROS-independent NET formation. In addition, transient bacteraemia due to tooth brushing indicates the possibility that citrullinated peptides and/or proteins from periodontium regularly enter the blood circulation. In this way, the mucosal firewall is evaded and the systemic immune response against citrullinated peptides and/or proteins is facilitated. However, the role of swallowed PD-derived sludge for the induction of oral tolerance remains to be established. We hypothesize (I) PD-driven endotoxemia may increase the host responsiveness to autoantigens via TLR4 activation and (II) this participates in development and propagation of RA (III) circulating PD-derived bacterial DNA is taken up by phagocytes, activates TLR9, and thus increases the responsiveness to autoantigens.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Endotoxemia/imunologia , Periodontite/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Aggregatibacter actinomycetemcomitans/enzimologia , Aggregatibacter actinomycetemcomitans/genética , Artrite Reumatoide/microbiologia , Autoantígenos/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Citrulinação/imunologia , Citrulina/metabolismo , DNA Bacteriano/imunologia , DNA Bacteriano/metabolismo , Endotoxemia/microbiologia , Armadilhas Extracelulares/enzimologia , Armadilhas Extracelulares/imunologia , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Humanos , Neutrófilos/enzimologia , Neutrófilos/imunologia , Periodontite/microbiologia , Periodonto/citologia , Periodonto/imunologia , Periodonto/metabolismo , Periodonto/microbiologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética , Desiminases de Arginina em Proteínas/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo
19.
Acta Reumatol Port ; 43(3): 239-240, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30414375

RESUMO

The presence of a biofilm-associated gingival inflammation in patients having juvenile systemic lupus erythematosus (jSLE) might function as a reservoir of anaerobic Gram-negative bacteria such as Porphyromonas gingivalis. Porphyromonas gingivalis also express functional endogenous peptidylarginine deiminase (PAD) enzymes, which catalyzes a citrullination reaction that can lead to formation of citrullinated peptides. We analyzed 30 patients with jSLE (16.2±1.5 years-old) and 29 healthy controls (15.4±2.3 years old jSLE was diagnosed according to the criteria of the American College of Rheumatology. The individuals underwent a full-mouth periodontal examination and were diagnosed with biofilm-associated gingival inflammation. Blood samples were taken and serum levels anti-PPAD IgG were determined by ELISA, and the results were expressed in arbitrary units (AU). The presence of Porphyromonas gingivalis was confirmed using the checkerboard DNA-DNA hybridization. Porphyromonas gingivalis was detected in both groups without any significant difference between them (p=0.58). The median levels of anti-PPAD were 0.54 AU (0.40 - 0.77) in juvenile SLE patients and 0.48 AU (0.26 - 0.97) in the control group (p=0.95). This is the first study to report the presence of anti-PPAD antibodies in serum from jSLEpatient.


Assuntos
Anticorpos/sangue , Lúpus Eritematoso Sistêmico/sangue , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/imunologia , Adolescente , Humanos
20.
mBio ; 9(5)2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30377277

RESUMO

The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.


Assuntos
Evasão da Resposta Imune , Imunidade Inata/efeitos dos fármacos , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Fatores de Virulência/metabolismo , Adulto , Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Gengiva/química , Gengiva/microbiologia , Humanos , Masculino , Periodontite/patologia , Fagocitose/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Desiminases de Arginina em Proteínas/análise , Fatores de Virulência/análise
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