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1.
Mol Oral Microbiol ; 36(6): 316-326, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34569151

RESUMO

The main etiological agent of periodontitis is the anaerobic bacterium Porphyromonas gingivalis. Virulence of this pathogen is controlled by various mechanisms and executed by major virulence factors including the gingipain proteases, peptidylarginine deiminase (PPAD), and RagB, an outer membrane macromolecular transport component. Although the structures and functions of these proteins are well characterized, little is known about their posttranslational maturation. Here, we determined the phosphoproteome of P. gingivalis in which phosphorylated tyrosine residues constitute over 80% of all phosphoresidues. Multiple phosphotyrosines were found in gingipains, PPAD, and RagB. Although mutation of phosphorylated residues in PPAD and RagB had no effect on secretion or activity, site-directed mutagenesis showed that phosphorylation in hemagglutinin/adhesin domains of RgpA and Kgp, and in the catalytic domain of RgpB, had a strong influence on secretion, processing, and enzymatic activity. Moreover, preventing phosphorylation of one gingipain influenced the others, suggesting multiple phosphorylation-dependent pathways of gingipain maturation in P. gingivalis. Various candidate kinases including Ptk1 BY kinase and ubiquitous bacterial kinase 1 (UbK1) may be involved, but their contribution to gingipain processing and activation remains to be confirmed.


Assuntos
Porphyromonas gingivalis , Fatores de Virulência , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Composição de Bases , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Hemaglutininas/genética , Fosforilação , Filogenia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , RNA Ribossômico 16S , Análise de Sequência de DNA , Fatores de Virulência/genética
2.
Mol Oral Microbiol ; 36(5): 258-266, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34241965

RESUMO

Phosphorylation of proteins is a key component of bacterial signaling systems that can control important functions such as community development and virulence. We report here the identification of a Ubiquitous bacterial Kinase (UbK) family member, designated UbK1, in the anaerobic periodontal pathogen, Porphyromonas gingivalis. UbK1 contains conserved SPT/S, Hanks-type HxDxYR, EW, and Walker A motifs, and a mutation analysis established the Walker A domain and the Hanks-type domain as required for both autophosphorylation and transphosphorylation. UbK1 autophosphorylates on the proximal serine in the SPT/S domain as well as the tyrosine residue within the HxDxYR domain and the tyrosine residue immediately proximal, indicating both serine/threonine and tyrosine specificity. The orphan two-component system response regulator (RR) RprY was phosphorylated on Y41 in the receiver domain by UbK1. The ubk1 gene is essential in P. gingivalis; however, overexpression of UbK1 showed that UbK1-mediated phosphorylation of RprY functions predominantly to augment its properties as a transcriptional enhancer. These results establish that P. gingivalis possesses an active UbK kinase in addition to a previously described Bacterial Tyrosine family kinase. The RR RprY is identified as the first transcriptional regulator controlled by a UbK enzyme.


Assuntos
Porphyromonas gingivalis , Transdução de Sinais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosforilação , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Proteínas Tirosina Quinases/metabolismo , Virulência
3.
Biomolecules ; 11(6)2021 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-34204019

RESUMO

Recent studies support the hypothesis that microbes can seed some Alzheimer's disease (AD) cases, leading to inflammation and overproduction of amyloid peptides. Porphyromonas gingivalis (Pg) is a keystone pathogen of chronic periodontitis and has been identified as risk factor for the development and progression of AD. The present preliminary study aimed to quantify Pg abundance in neurodegenerative disease (ND) patients compared with neurologic patients without neurodegenerative disorders (no-ND) and healthy controls (HC) to determine possible association between Pg abundance and neurodegenerative process. Pg was quantified on DNA extracted from the oral samples of 49 patients and 29 HC by quantitative polymerase chain reaction (qPCR). Anti-Pg antibodies were also detected on patient serum samples by enzyme-linked immunosorbent assays (ELISA). The Pg abundance in the oral cavity was significantly different among groups (p = 0.004). It was higher in ND than no-ND (p = 0.010) and HC (p = 0.008). The Pg abundance was correlated with the antibodies (p = 0.001) with different slopes between ND and no-ND (p = 0.037). Pg abundance was not correlated with oral indices and comorbidities. These results extend our understanding of the association between oral pathogens and AD to other neurodegenerative processes, confirming the hypothesis that oral pathogens can induce an antibody systemic response, influencing the progression of the disease.


Assuntos
Anticorpos Antibacterianos/sangue , Boca/microbiologia , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/microbiologia , Porphyromonas gingivalis/metabolismo , Idoso , Idoso de 80 Anos ou mais , Infecções por Bacteroidaceae/sangue , Infecções por Bacteroidaceae/diagnóstico , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/diagnóstico , Projetos Piloto , Porphyromonas gingivalis/isolamento & purificação
4.
mBio ; 12(3): e0050221, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34182783

RESUMO

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.


Assuntos
Gengiva/citologia , Inflamação , Queratinócitos/imunologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/metabolismo , Ribonucleases/metabolismo , Transdução de Sinais , Animais , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Feminino , Cisteína Endopeptidases Gingipaínas , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/microbiologia , Porphyromonas gingivalis/fisiologia , Ribonucleases/genética , Ribonucleases/imunologia , Organismos Livres de Patógenos Específicos
5.
Arch Oral Biol ; 126: 105129, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33934042

RESUMO

OBJECTIVE: To determine if AP5055 drug, an inhibitor of CD36, prevents the increase in Porphyromonas gingivalis (P. gingivalis) mediated atherosclerosis in low-density lipoprotein receptor knockout (LDLR KO) mice by targeting CD36. METHODS: Male LDLR KO mice were infected with P. gingivalis by oral lavage to induce periodontal disease and fed a western diet to induce atherosclerosis. Mice were treated with the CD36 inhibitor, AP5055 (1 mg/kg), or vehicle (1% DMSO). Aortae were dissected and stained with oil red-O for morphometric analysis; blood/plasma was collected to determine markers of inflammation by cytokine array and cholesterol levels. P. gingivalis-induced bone loss in mandibles was assessed using micro-CT. P. gingivalis lipopolysaccharide stimulated nuclear factor-kappa B (NF-κB) activity was measured using a reporter gene (secreted alkaline phosphatase) assay in AP5055 treated or untreated RAW-Blue macrophages. RESULTS: Isolated aortae showed a significant decrease in lesion area in the AP5055 treated group as compared to the control group. Mechanistically, in vitro analysis demonstrated that AP5055 inhibited NF-κB activity. Cytokine array showed a decrease in the expression of pro-inflammatory cytokines and decreased levels of plasma cholesterol in AP5055 treated mice. Micro-CT measurements of bone loss were not significant between the two groups. CONCLUSION: CD36 inhibitor AP5055 abrogates atherosclerotic lesion burden associated with periodontal disease, accompanied by a reduction in markers of inflammation. These experiments may support the development of drugs targeting CD36 for human disease.


Assuntos
Aterosclerose , Porphyromonas gingivalis , Animais , Antígenos CD36 , Lipopolissacarídeos , Masculino , Camundongos , NF-kappa B/metabolismo , Porphyromonas gingivalis/metabolismo
6.
FEBS Lett ; 595(11): 1604-1612, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792027

RESUMO

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.


Assuntos
Cisteína Endopeptidases Gingipaínas/farmacologia , Glicoproteínas da Membrana de Plaquetas/genética , Porphyromonas gingivalis/metabolismo , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética , Fatores de Virulência/farmacologia , Células A549 , Aderência Bacteriana/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Fímbrias Bacterianas/química , Regulação da Expressão Gênica , Cisteína Endopeptidases Gingipaínas/deficiência , Cisteína Endopeptidases Gingipaínas/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Lipopolissacarídeos/farmacologia , Modelos Biológicos , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , Alvéolos Pulmonares/microbiologia , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Virulência/deficiência , Fatores de Virulência/genética
7.
J Bacteriol ; 203(10)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33685973

RESUMO

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCE Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Mutação , Pigmentação/genética , Porphyromonas gingivalis/genética
8.
Biochim Biophys Acta Proteins Proteom ; 1869(7): 140652, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33746063

RESUMO

Methionine-γ-lyase (MGL) is a pyridoxal-5'-phosphate dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Here we report experimental kinetic data and density functional theory (DFT) computational data for the γ-elimination reaction of L-Met and several other substrate analogues by a recombinant MGL from P. gingivalis (MGL_Pg). UV-Visible spectrophotometry experiments revealed a heavily populated species with maximum absorbance at 478 nm during steady-state catalysis of L-Met, L-ethionine, L-methionine sulfone and L-homoserine, which we assign to a late crotonate intermediate formed after the γ-cleavage step in the reaction and thus common to all substrates. A more red-shifted (498 nm) species was observed during the reaction of L-homoserine lactone, which we assign to an early quinonoid intermediate with the aid of time-dependent self-consistent field calculations. Significant differences in both binding and the rate of turnover were observed for the substrates. MGL_Pg's highest catalytic efficiency was recorded for L-vinylglycine (kcat/Km = 6455 s-1 M-1), exceeding that of L-Met (kcat/Km = 4211 s-1 M-1), while L-Met sulfone displayed the largest turnover number (kcat = 1638 min-1). A direct correlation between experimental kcat values and DFT-calculated γ-cleavage Gibbs activation energies was identified for the various substrates. In light of these data, we propose that the γ-cleavage step in the catalytic reaction pathway is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.


Assuntos
Liases de Carbono-Enxofre/metabolismo , Metionina/metabolismo , Liases de Carbono-Enxofre/química , Catálise , Cisteína/metabolismo , Cinética , Metionina/análogos & derivados , Metionina/química , Porphyromonas gingivalis/metabolismo , Espectrofotometria/métodos , Especificidade por Substrato
9.
Mol Oral Microbiol ; 36(3): 172-181, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33715305

RESUMO

Programmed death-ligand-1 (PD-L1) is a ligand for programmed death receptor (PD-1) that plays a major role in cell-mediated immune response; it regulates T-cell activation and regulates survival and functions of activated T cells. Expression of PD-L1 can induce chronic inflammation and activate mechanisms of immune evasion. PD-L1 is expressed in most of human carcinomas. Porphyromonas gingivalis (P. gingivalis) is a major keystone pathogen in periodontitis that invade host cells and disposes a variety of virulence factors. The aim of the present study was to clarify the signaling pathway of P. gingivalis molecules that induce PD-L1 up-regulation in colon carcinoma cells. Additionally, it was investigated which components of P. gingivalis are responsible for PD-L1 induction. Colon cancer cells (CL-11) were stimulated with total membrane (TM) fractions, peptidoglycans (PDGs) and viable P. gingivalis bacteria. Seven signaling molecule inhibitors were used: receptor-interacting serine/threonine-protein kinase 2 (RIP2) tyrosine kinase inhibitor, nucleotide-binding oligomerization domain (NOD)-like receptor 1&2 inhibitor, NOD-like receptor, nuclear factor kappa B inhibitor, c-Jun N-terminal kinases inhibitor, mitogen-activated protein/extracellular signal-regulated kinase inhibitor, mitogen activated kinase (MAPK) inhibitor. PD-L1 protein expression was examined by western blot analysis and quantitative real time PCR. It was demonstrated that the TM fraction and PDG induced up-regulation of PD-L1 expression in colon cancer cells. In conclusion, the results of this study suggest that PDG of P. gingivalis plays a major role in PD-L1 up-regulation in colon cancer cells. In addition, the mechanism of PD-L1 up-regulation depends on NOD 1 and NOD 2 and involves activation of RIP2 and MAPK signaling pathways.


Assuntos
Carcinoma , Neoplasias do Colo , Antígeno B7-H1/metabolismo , Humanos , Porphyromonas gingivalis/metabolismo , Regulação para Cima
10.
J Biol Chem ; 296: 100574, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33757767

RESUMO

PorX/PorY is a two-component system (TCS) of Porphyromonas gingivalis that governs transcription of numerous genes including those encoding a type IX secretion system (T9SS) for gingipain secretion and heme accumulation. Here, an in vitro analysis showed that the response regulator PorX specifically bound to two regions in the promoter of porT, a known PorX-regulated T9SS gene, thus demonstrating that PorX/PorY can directly regulate specific target genes. A truncated PorX protein containing the N-terminal receiver and effector domains retained a wild-type ability in both transcription regulation and heme accumulation, ruling out the role of the C-terminal ALP domain in gene regulation. The PorX/PorY system was the only TCS essential for heme accumulation and concomitantly responded to hemin to stimulate transcription of several known PorX-dependent genes in a concentration-dependent manner. We found that PorX/PorY activated the sigH gene, which encodes a sigma factor known for P. gingivalis adaptation to hydrogen peroxide (H2O2). Consistently, both ΔporX and ΔsigH mutants were susceptible to H2O2, suggesting a PorX/PorY-σH regulatory cascade to confer resistance to oxidative stress. Furthermore, the ΔporX mutant became susceptible to high hemin levels that could induce oxidative stress. Therefore, a possible reason why hemin activates PorX/PorY is to confer resistance to hemin-induced oxidative stress. We also demonstrated that PorX/PorY was essential for P. gingivalis virulence because the ΔporX mutant was avirulent in a mouse model. Specifically, this TCS was required for the repression of proinflammatory cytokines secreted by dendritic cells and T cells in the P. gingivalis-infected mice.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Camundongos , Mutação , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/fisiologia , Processamento de Proteína Pós-Traducional , Fatores de Virulência/genética
11.
Int J Mol Sci ; 22(5)2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33668119

RESUMO

Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Heme/metabolismo , Porphyromonas gingivalis/metabolismo , Humanos , Ligação Proteica
12.
J Periodontal Res ; 56(3): 523-534, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33533512

RESUMO

BACKGROUND/OBJECTIVES: Iron homeostasis plays a crucial role in the combat against pathogen invasion. Ferrous iron can trigger generous production of reactive oxygen species (ROS) by Fenton reaction. Nuclear receptor coactivator 4 (NCOA4), a selective cargo receptor to deliver ferritin to lysosome, may trigger release of ferritin-bound iron into the cytosol. The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis, and its role in promoting the periodontal inflammation. METHODS: Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues, while real-time quantitative PCR (qPCR) was utilized to detect mRNA transcription. Periodontal ligament fibroblasts (PDLFs) were isolated and infected with Porphyromonas gingivalis. The mRNA transcription and protein expression of genes involved in the iron metabolism, including NCOA4, transferrin receptor 1 (TFR1), and ferroportin (SLC40A1) were detected by qPCR and western blot. Levels of labile iron pool and ROS production were detected by flow cytometry and confocal endoscopy. Small interference RNA was utilized to knock down NCOA4. RESULTS: Elevated expression of NCOA4, ferritin heavy chain, and light chain were observed in the diseased periodontal tissues. P. gingivalis infection promoted expression of TFR1, NCOA4, and microtubule-associated protein 1-light chain 3 B (LC3B), enhanced levels of intracellular labile iron pool and ROS production. NCOA4 knockdown reduced ROS generation in PDLFs in response to P. gingivalis and mitigated production of pro-inflammatory monocyte chemoattractant protein-1 and interleukin 6. P. gingivalis triggered activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling pathway. In addition, inhibitors of JNK, SP600125, and inhibitors of p38, SB203580 blocked NCOA4 transcription. CONCLUSION: NCOA4-ferritinophagy participated in the progress of periodontitis progression. P. gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS. These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis.


Assuntos
Ferritinas , Periodontite , Humanos , Ferro/metabolismo , Lisossomos/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/metabolismo
13.
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
14.
Adipocyte ; 10(1): 28-37, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33393852

RESUMO

Obesity is an important public-health problem worldwide. This study aimed to determine effects of porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on adipocytes injuries and explore associated mechanisms. Adipocytes were isolated from SD rats. pLVX-XBP1 (XBP1 over-expression) and pLVX-XBP1-RNAi (silencing XBP1) were structured and transfected into adipocytes. All adipocytes were divided into pLVX-NC, pLVX-XBP1, pLVX-NC+Pg-LPS and pLVX-XBP1+ Pg-LPS group. Oil-Red O staining was employed to identify isolated adipocytes. Quantitative real-time PCR (qRT-PCR) was used to examine gene transcription of IL-6, TNF-α, leptin, adiponectin. Western blotting was used to detect Bax and caspase-3 expression. Adipocytes were successfully isolated and identified with Oil-Red O staining. Both XBP1 mimic and XBP1 RNAi were effectively transfected into adipocytes with higher expressing efficacy. XBP1 over-expression significantly aggravated Pg-LPS induced inflammatory response compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS significantly enhanced leptin and inhibited adiponectin expression by up-regulating XBP1 expression (p<0.05). XBP1 silence significantly alleviated Pg-LPS induced inflammatory response and reduced leptin, enhanced adiponectin expression in Pg-LPS treated adipocytes compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS induced apoptosis of adipocytes by enhancing XBP1 expression and modulating Bcl-2/Bax pathway associated molecules. In conclusion, Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) induces adipocytes injuries through modulating XBP1 expression and initialling mitochondria-mediated apoptosis.


Assuntos
Adipócitos/metabolismo , Lipopolissacarídeos/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , China , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidade , Ratos , Ratos Sprague-Dawley , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/fisiologia
15.
J Cell Physiol ; 236(8): 5715-5724, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33400284

RESUMO

Periodontal ligament fibroblasts (PdLFs) are an elongated cell type in the periodontium with matrix and bone regulatory functions which become abnormal in periodontal disease (PD). Here we found that the normally elongated and oriented PdLF nucleus becomes rounded and loses orientation in a mouse model of PD. Using in vitro micropatterning of cultured primary PdLF cell shape, we show that PdLF elongation correlates with nuclear elongation and the presence of thicker, contractile F-actin fibers. The rounded nuclei in mouse PD models in vivo are, therefore, indicative of reduced actomyosin tension. Inhibiting actomyosin contractility by inhibiting myosin light chain kinase, Rho kinase or myosin ATPase activity, in cultured PdLFs each consistently reduced messenger RNA levels of bone regulatory protein osteoprotegerin (OPG). Infection of cultured PdLFs with two different types of periodontal bacteria (Porphyromonas gingivalis and Fusobacterium nucleatum) failed to recapitulate the observed nuclear rounding in vivo, upregulated nonmuscle myosin II phosphorylation and downregulated OPG. Collectively, our results add support to the hypothesis that PdLF contractility becomes decreased and contributes to disease progression in PD.


Assuntos
Actomiosina/metabolismo , Fibroblastos/metabolismo , Osteoprotegerina/metabolismo , Ligamento Periodontal/efeitos dos fármacos , Animais , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/metabolismo
16.
Nat Microbiol ; 6(2): 221-233, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432152

RESUMO

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.


Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos/química , Flavobacterium/fisiologia , Porphyromonas gingivalis/fisiologia , Microscopia Crioeletrônica , Flavobacterium/metabolismo , Movimento , Periplasma/metabolismo , Porphyromonas gingivalis/metabolismo , Domínios Proteicos , Multimerização Proteica , Prótons , Imagem Individual de Molécula
17.
Trends Microbiol ; 29(1): 54-64, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33071035

RESUMO

Proteases are critical virulence determinants of Porphyromonas gingivalis, an emerging Alzheimer's disease, cancer, and arthritis pathogen and established agent of periodontitis. Transposon sequencing has been employed to define the core essential genome of this bacterium and genes conditionally essential in multiple environments - abscess formation; epithelial colonization; and cigarette smoke toxin exposure; as well as to elucidate genes required for iron acquisition and a functional type 9 secretion system. Validated and predicted protein catabolism genes identified include a combination of established virulence factors and a larger set of seemingly more mundane proteolytic genes. The functions and relevance of genes that share essentiality in multiple disease-relevant conditions are examined. These common stress-related genes may represent particularly attractive therapeutic targets for the control of P. gingivalis infections.


Assuntos
Proteínas de Bactérias/metabolismo , Porphyromonas gingivalis/metabolismo , Animais , Proteínas de Bactérias/genética , Infecções por Bacteroidaceae/microbiologia , Genes Essenciais , Humanos , Porphyromonas gingivalis/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
18.
Photochem Photobiol ; 97(2): 377-384, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32959424

RESUMO

In vitro experiments confirmed that antibacterial photodynamic treatment (aPDT) inactivates periodontal pathogens. However, more effective sterilization is needed in the complex oral environment. This study tested whether dihydroartemisinin (DHA) enhanced the photokilling effect of aPDT on Porphyromonas gingivalis (P. gingivalis) in planktonic and biofilm states. aPDT combining toluidine blue O (TBO) with 630 nm red light was performed on bacterial suspensions and biofilms in vitro with different final concentrations of DHA (10, 20 and 40 µg mL-1 ). The sensitization mechanism was preliminarily investigated by uptake experiments. The above experiments were repeated with different incubation times (30, 60, 120 s). Porphyromonas gingivalis biofilms exhibited significantly higher resistance to aPDT than P. gingivalis in suspension under the same experimental parameters. DHA alone had no cytotoxic effect on P. gingivalis with or without light irradiation. In either bacterial suspensions or biofilms, DHA concentration-dependently enhanced the photokilling effect of aPDT and increased TBO uptake by P. gingivalis. Prolonged incubation time enhanced the photokilling efficiency of aPDT until cellular TBO uptake reached saturation. DHA can enhance aPDT activity against P. gingivalis in planktonic and biofilm states. DHA also accelerated TBO uptake, reducing incubation time.


Assuntos
Antibacterianos/farmacologia , Artemisininas/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Cloreto de Tolônio/farmacologia , Biofilmes/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Fármacos Fotossensibilizantes/metabolismo , Porphyromonas gingivalis/metabolismo , Cloreto de Tolônio/metabolismo
19.
Infect Immun ; 89(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33257533

RESUMO

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis, an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-ß) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis-associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Apresentação Cruzada/fisiologia , Células Dendríticas/metabolismo , Imunidade Inata/fisiologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidade , Variação Genética , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Periodonto/microbiologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/imunologia
20.
FEBS J ; 288(5): 1479-1495, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32681704

RESUMO

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface-expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg-induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild-type (W83) but not gingipain-deficient (ΔK/R-ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM-1 and VE-cadherin compared to infection with ΔK/R-ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM-1 or VE-cadherin were systemically infected with W83 or ΔK/R-ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain-dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM-1 and VE-cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease.


Assuntos
Infecções por Bacteroidaceae/etiologia , Cardiomegalia/etiologia , Edema/etiologia , Células Endoteliais/efeitos dos fármacos , Cisteína Endopeptidases Gingipaínas/toxicidade , Porphyromonas gingivalis/patogenicidade , Animais , Animais Geneticamente Modificados , Antígenos CD/genética , Antígenos CD/metabolismo , Infecções por Bacteroidaceae/genética , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/patologia , Caderinas/genética , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Edema/genética , Edema/metabolismo , Edema/patologia , Embrião não Mamífero , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Angiofluoresceinografia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Cisteína Endopeptidases Gingipaínas/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva/efeitos dos fármacos , Larva/microbiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/metabolismo , Cultura Primária de Células , Transdução de Sinais , Peixe-Zebra
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