RESUMEN

Porphyromonas gingivalis has been demonstrated to have the strongest association with periodontitis. Within the host, P. gingivalis relies on acquiring iron and heme through the aggregation and lysis of erythrocytes, which are important factors in the growth and virulence of P. gingivalis. Additionally, the excess obtained heme is deposited on the surface of P. gingivalis, protecting the cells from oxidative damage. Based on these biological properties of the interaction between P. gingivalis and erythrocytes, this study developed an erythrocyte membrane nanovesicle loaded with gallium porphyrins to mimic erythrocytes. The nanovesicle can target and adhere with P. gingivalis precisely, being lysed and utilized by P. gingivalis as erythrocytes. Ingested gallium porphyrin replaces iron porphyrin in P. gingivalis, causing intracellular metabolic disruption. Deposited porphyrin generates a large amount of reactive oxygen species (ROS) under blue light, causing oxidative damage, and its lethality is enhanced by bacterial metabolic disruption, synergistically killing P. gingivalis. Our results demonstrate that this strategy can target and inhibit P. gingivalis, reduce its invasion of epithelial cells, and alleviate the progression of periodontitis.

Asunto(s)

Eritrocitos , Periodontitis , Porfirinas , Porphyromonas gingivalis , Porphyromonas gingivalis/efectos de los fármacos , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/química , Periodontitis/microbiología , Periodontitis/tratamiento farmacológico , Periodontitis/patología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Porfirinas/química , Porfirinas/farmacología , Animales , Especies Reactivas de Oxígeno/metabolismo , Galio/química , Galio/farmacología , Ratones , Antibacterianos/farmacología , Antibacterianos/química , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología

RESUMEN

Periodontitis is a critical risk factor for the occurrence and development of diabetes. Porphyromonas gingivalis may participate in insulin resistance (IR) caused by periodontal inflammation, but the functional role and specific mechanisms of P. gingivalis in IR remain unclear. In the present study, clinical samples were analysed to determine the statistical correlation between P. gingivalis and IR occurrence. Through culturing of hepatocytes, myocytes, and adipocytes, and feeding mice P. gingivalis orally, the functional correlation between P. gingivalis and IR occurrence was further studied both in vitro and in vivo. Clinical data suggested that the amount of P. gingivalis isolated was correlated with the Homeostatic Model Assessment for IR score. In vitro studies suggested that coculture with P. gingivalis decreased glucose uptake and insulin receptor (INSR) protein expression in hepatocytes, myocytes, and adipocytes. Mice fed P. gingivalis tended to undergo IR. P. gingivalis was detectable in the liver, skeletal muscle, and adipose tissue of experimental mice. The distribution sites of gingipain coincided with the downregulation of INSR. Gingipain proteolysed the functional insulin-binding region of INSR. Coculture with P. gingivalis significantly decreased the INSR-insulin binding ability. Knocking out gingipain from P. gingivalis alleviated the negative effects of P. gingivalis on IR in vivo. Taken together, these findings indicate that distantly migrated P. gingivalis may directly proteolytically degrade INSR through gingipain, thereby leading to IR. The results provide a new strategy for preventing diabetes by targeting periodontal pathogens and provide new ideas for exploring novel mechanisms by which periodontal inflammation affects the systemic metabolic state.

Asunto(s)

Cisteína-Endopeptidasas Gingipaínas , Resistencia a la Insulina , Porphyromonas gingivalis , Receptor de Insulina , Porphyromonas gingivalis/metabolismo , Receptor de Insulina/metabolismo , Animales , Ratones , Cisteína-Endopeptidasas Gingipaínas/metabolismo , Humanos , Masculino , Adhesinas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteolisis , Femenino , Adipocitos/metabolismo , Periodontitis/microbiología , Técnicas de Cocultivo

RESUMEN

Background: Trimethylamine-N-oxide (TMAO) is produced by hepatic flavin-containing monooxygenase 3 (FMO3) from trimethylamine (TMA). High TMAO level is a biomarker of cardiovascular diseases and metabolic disorders, and it also affects periodontitis through interactions with the gastrointestinal microbiome. While recent findings indicate that periodontitis may alter systemic TMAO levels, the specific mechanisms linking these changes and particular oral pathogens require further clarification. Methods: In this study, we established a C57BL/6J male mouse model by orally administering Porphyromonas gingivalis (P. gingivalis, Pg), Fusobacterium nucleatum (F. nucleatum, Fn), Streptococcus mutans (S. mutans, Sm) and PBS was used as a control. We conducted LC-MS/MS analysis to quantify the concentrations of TMAO and its precursors in the plasma and cecal contents of mice. The diversity and composition of the gut microbiome were analyzed using 16S rRNA sequencing. TMAO-related lipid metabolism and enzymes in the intestines and liver were assessed by qPCR and ELISA methods. We further explored the effect of Pg on FMO3 expression and lipid molecules in HepG2 cells by stimulating the cells with Pg-LPS in vitro. Results: The three oral pathogenic bacteria were orally administered to the mice for 5 weeks. The Pg group showed a marked increase in plasma TMAO, betaine, and creatinine levels, whereas no significant differences were observed in the gut TMAO level among the four groups. Further analysis showed similar diversity and composition in the gut microbiomes of both the Pg and Fn groups, which were different from the Sm and control groups. The profiles of TMA-TMAO pathway-related genera and gut enzymes were not significantly different among all groups. The Pg group showed significantly higher liver FMO3 levels and elevated lipid factors (IL-6, TG, TC, and NEFA) in contrast to the other groups. In vitro experiments confirmed that stimulation of HepG2 cells with Pg-LPS upregulated the expression of FMO3 and increased the lipid factors TC, TG, and IL-6. Conclusion: This study conclusively demonstrates that Pg, compared to Fn and Sm, plays a critical role in elevating plasma TMAO levels and significantly influences the TMA-TMAO pathway, primarily by modulating the expression of hepatic FMO3 and directly impacting hepatic lipid metabolism.

Asunto(s)

Microbioma Gastrointestinal , Metilaminas , Ratones Endogámicos C57BL , Oxigenasas , Porphyromonas gingivalis , Animales , Masculino , Metilaminas/metabolismo , Metilaminas/sangre , Humanos , Ratones , Oxigenasas/metabolismo , Porphyromonas gingivalis/metabolismo , Fusobacterium nucleatum/metabolismo , Redes y Vías Metabólicas , Células Hep G2 , Metabolismo de los Lípidos , Modelos Animales de Enfermedad , Periodontitis/microbiología , Periodontitis/metabolismo , Hígado/metabolismo , ARN Ribosómico 16S/genética , Espectrometría de Masas en Tándem , Boca/microbiología

RESUMEN

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Asunto(s)

Lipopolisacáridos , Porphyromonas gingivalis , Transducción de Señal , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Lipopolisacáridos/metabolismo , Factores de Virulencia/metabolismo , Regulación Bacteriana de la Expresión Génica , Metabolismo Energético , Fosfatos de Dinucleósidos/metabolismo , Ácidos Grasos/metabolismo , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética

RESUMEN

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Asunto(s)

Proteínas Bacterianas , Sistemas de Secreción Bacterianos , Porphyromonas gingivalis , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/metabolismo , Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/química , Modelos Moleculares , Cristalografía por Rayos X , Secuencia de Aminoácidos , Señales de Clasificación de Proteína , Dominios Proteicos , Bacteroidetes/metabolismo , Bacteroidetes/genética , Tannerella forsythia/metabolismo , Tannerella forsythia/genética , Tannerella forsythia/química , Relación Estructura-Actividad , Conformación Proteica

RESUMEN

Introduction: Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein. Methods: Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium. Results: Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin. Conclusions: We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.

Asunto(s)

Proteínas Bacterianas , Hemo , Porphyromonas endodontalis , Porphyromonas gingivalis , Tannerella forsythia , Hemo/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Tannerella forsythia/metabolismo , Tannerella forsythia/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Porphyromonas endodontalis/metabolismo , Porphyromonas endodontalis/genética , Humanos , Regulación Bacteriana de la Expresión Génica , Unión Proteica , Hierro/metabolismo

RESUMEN

Porphyromonas gingivalis is a nonmotile, obligate anaerobic, Gram-negative bacterium known for its association with periodontal disease and its involvement in systemic diseases such as atherosclerosis, cardiovascular disease, colon cancer, and Alzheimer's disease. This bacterium produces several virulence factors, including capsules, fimbriae, lipopolysaccharides, proteolytic enzymes, and hemagglutinins. A comparative genomic analysis revealed the open pangenome of P. gingivalis and identified complete type IV secretion systems in strain KCOM2805 and almost complete type VI secretion systems in strains KCOM2798 and ATCC49417, which is a new discovery as previous studies did not find the proteins involved in secretion systems IV and VI. Conservation of some virulence factors between different strains was observed, regardless of their genetic diversity and origin. In addition, we performed for the first time a reconstruction analysis of the gene regulatory network, identifying transcription factors and proteins involved in the regulatory mechanisms of bacterial pathogenesis. In particular, QseB regulates the expression of hemagglutinin and arginine deaminase, while Rex may suppress the release of gingipain through interactions with PorV and the formatum/nitrate transporter. Our study highlights the central role of conserved virulence factors and regulatory pathways, particularly QseB and Rex, in P. gingivalis and provides insights into potential therapeutic targets.

Asunto(s)

Redes Reguladoras de Genes , Genoma Bacteriano , Redes y Vías Metabólicas , Porphyromonas gingivalis , Factores de Virulencia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidad , Factores de Virulencia/genética , Redes y Vías Metabólicas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Regulación Bacteriana de la Expresión Génica

RESUMEN

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1ß and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

Asunto(s)

Barrera Hematoencefálica , Vesículas Extracelulares , Encía , Periodontitis , Porphyromonas gingivalis , Barrera Hematoencefálica/metabolismo , Animales , Humanos , Ratones , Vesículas Extracelulares/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidad , Periodontitis/microbiología , Periodontitis/metabolismo , Periodontitis/patología , Encía/metabolismo , Encía/microbiología , Ratones Endogámicos C57BL , Masculino , Exosomas/metabolismo , Femenino , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/metabolismo

RESUMEN

OBJECTIVES: Periodontitis, commonly associated with Porphyromonas gingivalis (Pg), involves intricate alterations of oral intercellular interactions, in which extracellular vesicles (EVs) play a pivotal role. The understanding of the miRNA profiles in the EVs derived from Pg-infected cells (Pg-EVs) remains incomplete despite acknowledging their importance in intercellular communication during periodontitis. Therefore, our objective was to identify and characterize the miRNAs enriched in Pg-EVs. METHODS: Microarray analysis was conducted to examine the miRNA profiles in the EVs derived from Pg-infected THP-1 cells. We compared the identified miRNAs with those upregulated in the EVs after stimulation with LPS. Additionally, we explored how inhibiting TLR signaling during Pg infection affects the transcription of specific miRNAs. We investigated the unique sequence motifs specific to the miRNAs concentrated in Pg-EVs. RESULTS: The levels of eleven miRNAs, including miR-155, were increased in Pg-EVs compared with those elevated after LPS stimulation. The Pg-induced miR-155 upregulation via TLR2 but not TLR4 signaling suggests the influence of TLR signaling on the miRNA composition of EVs. Furthermore, the miRNAs upregulated in Pg-EVs contained AGAGGG and GRGGSGC sequence motifs. CONCLUSIONS: Our findings demonstrate that Pg-induced alterations in EV-containing miRNA composition occur in a TLR4-independent manner. Notably, the concentrated miRNAs in Pg-EVs harbor specific motifs with a high G + C content within their sequences. The upregulation of specific miRNAs in EVs under infectious conditions suggests the influence of both innate immune receptor signals and miRNA sequence characteristics.

Asunto(s)

Vesículas Extracelulares , MicroARNs , Porphyromonas gingivalis , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , MicroARNs/metabolismo , MicroARNs/genética , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Humanos , Transducción de Señal , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/genética , Lipopolisacáridos/farmacología , Periodontitis/microbiología , Periodontitis/metabolismo , Periodontitis/genética , Regulación hacia Arriba , Análisis por Micromatrices

RESUMEN

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

Asunto(s)

Resorción Ósea , Periodontitis , Receptor EphB2 , Receptor EphB4 , Animales , Ratas , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/microbiología , Osteoclastos/metabolismo , Periodontitis/microbiología , Porphyromonas gingivalis/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Transducción de Señal , Receptor EphB2/metabolismo , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología

RESUMEN

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Asunto(s)

Proteínas Bacterianas , Flavobacterium , Proteínas Bacterianas/metabolismo , Flavobacterium/metabolismo , Bacteroidetes/metabolismo , Actividad Motora , Sistemas de Secreción Bacterianos/genética , Porphyromonas gingivalis/metabolismo

RESUMEN

OBJECTIVE: The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid ß (Aß) metabolites in linking periodontitis to Alzheimer's disease (AD). BACKGROUND: Aß is one of the main pathological features of AD, and few studies have discussed changes in its expression in peripheral tissues or analyzed the relationship between the peripheral imbalance of Aß production and clearance. METHODS: A murine model of periodontitis was established by oral infection with Porphyromonas gingivalis (P. gingivalis). Micro-computed tomography (Micro-CT) was used to observe the destruction of the alveolar bone. Nested quantitative polymerase chain reaction (qPCR) was used to measure small quantities of P.gingivalis DNA in different tissues. Behavioral experiments were performed to measure cognitive function in the mice. The mRNA levels of TNF-α, IL-6, IL-8, RANKL, OPG, APP695, APP751, APP770, and BACE1 in the gingival tissues or cortex were detected by RT-PCR. The levels of Aß1-40 and Aß1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. RESULTS: P. gingivalis oral infection was found to cause alveolar bone resorption and impaired learning and memory. P.gingivalis DNA was detected in the gingiva, blood and cortex of the P.gingivalis group by nested qPCR (p < .05). The mRNA expression of TNF-α, IL-6, IL-8, RANKL/OPG, and BACE1 in the gingival tissue was significantly higher than that in the control group (p < .05). Similarly, upregulated mRNA levels of APP695 and APP770 were observed in the gingival tissuses and cortex of the P. gingivalis group (p < .05). The levels of Aß1-40 and Aß1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). CONCLUSION: P. gingivalis can directly invade the brain via hematogenous infection. The invasion of P. gingivalis could trigger an immune response and lead to an imbalance between Aß production and clearance in peripheral tissues, which may trigger an abnormal Aß metabolite in the brain, resulting in the occurrence and development of AD.

Asunto(s)

Pérdida de Hueso Alveolar , Periodontitis , Ratones , Animales , Precursor de Proteína beta-Amiloide/genética , Porphyromonas gingivalis/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/metabolismo , Factor de Necrosis Tumoral alfa , Modelos Animales de Enfermedad , Microtomografía por Rayos X , Interleucina-6 , Interleucina-8 , Ácido Aspártico Endopeptidasas , Periodontitis/metabolismo , ARN Mensajero/análisis , ADN

RESUMEN

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

Asunto(s)

Pérdida de Hueso Alveolar , Porphyromonas gingivalis , Masculino , Humanos , Animales , Ratones , Porphyromonas gingivalis/metabolismo , Claudina-1/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Claudina-2/metabolismo , Ratones Endogámicos C57BL , Cadherinas/metabolismo , Envejecimiento , Conexinas/metabolismo

RESUMEN

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability.

Asunto(s)

Adhesinas Bacterianas , Cisteína-Endopeptidasas Gingipaínas , Hemo , Niacinamida , Periodontitis , Porphyromonas gingivalis , Porphyromonas gingivalis/efectos de los fármacos , Porphyromonas gingivalis/patogenicidad , Porphyromonas gingivalis/metabolismo , Virulencia/efectos de los fármacos , Animales , Niacinamida/farmacología , Hemo/metabolismo , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/efectos de los fármacos , Ratones , Periodontitis/microbiología , Periodontitis/prevención & control , Hemólisis/efectos de los fármacos , Pérdida de Hueso Alveolar/prevención & control , Pérdida de Hueso Alveolar/microbiología , Modelos Animales de Enfermedad , Antibacterianos/farmacología , Cisteína Endopeptidasas/metabolismo , Hemaglutinación/efectos de los fármacos , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/tratamiento farmacológico , Infecciones por Bacteroidaceae/prevención & control , Humanos

RESUMEN

Oral microbiome dysbiosis mediates chronic periodontal disease, gut microbial dysbiosis, and mucosal barrier disfunction that leads to steatohepatitis via the enterohepatic circulation. Improving this dysbiosis towards health may improve liver disease. Treatment with antibiotics and probiotics have been used to modulate the microbial, immunological, and clinical landscape of periodontal disease with some success. The aim of the present investigation was to evaluate the potential for nisin, an antimicrobial peptide produced by Lactococcus lactis, to counteract the periodontitis-associated gut dysbiosis and to modulate the glycolipid-metabolism and inflammation in the liver. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum, were administrated topically onto the oral cavity to establish polymicrobial periodontal disease in mice. In the context of disease, nisin treatment significantly shifted the microbiome towards a new composition, commensurate with health while preventing the harmful inflammation in the small intestine concomitant with decreased villi structural integrity, and heightened hepatic exposure to bacteria and lipid and malondialdehyde accumulation in the liver. Validation with RNA Seq analyses, confirmed the significant infection-related alteration of several genes involved in mitochondrial dysregulation, oxidative phosphorylation, and metal/iron binding and their restitution following nisin treatment. In support of these in vivo findings indicating that periodontopathogens induce gastrointestinal and liver distant organ lesions, human autopsy specimens demonstrated a correlation between tooth loss and severity of liver disease. Nisin's ability to shift the gut and liver microbiome towards a new state commensurate with health while mitigating enteritis, represents a novel approach to treating NAFLD-steatohepatitis-associated periodontal disease.

Asunto(s)

Bacteriocinas , Nisina , Enfermedad del Hígado Graso no Alcohólico , Enfermedades Periodontales , Ratones , Humanos , Animales , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Nisina/farmacología , Nisina/metabolismo , Disbiosis , Enfermedades Periodontales/microbiología , Porphyromonas gingivalis/metabolismo , Inflamación/complicaciones , Estrés Oxidativo

RESUMEN

Porphyromonas gingivalis strains exhibit different phenotypes in vitro, different virulence potential in animal models, and different associations with human diseases, with strains classified as virulent/more virulent (e.g., A7436 and W83) or as less virulent/avirulent (e.g., ATCC 33277). In this study, we comparatively analyzed the A7436 and ATCC 33277 strains to better understand their variability. Global gene expression analysis in response to heme and iron limitation revealed more pronounced differences in the A7436 than in the ATCC 33277 strain; however, in both strains, the largest changes were observed in genes encoding hypothetical proteins, genes whose products participate in energy metabolism, and in genes encoding proteins engaged in transport and binding proteins. Our results confirmed that variability between P. gingivalis strains is due to differences in the arrangement of their genomes. Analysis of gene expression of heme acquisition systems demonstrated that not only the availability of iron and heme in the external environment but also the ability to store iron intracellularly can influence the P. gingivalis phenotype. Therefore, we assume that differences in virulence potential may also be due to differences in the production of systems involved in iron and heme acquisition, mainly the Hmu system. In addition, our study showed that hemoglobin, in a concentration-dependent manner, differentially influences the virulence potential of P. gingivalis strains. We conclude that iron and heme homeostasis may add to the variability observed between P. gingivalis strains. IMPORTANCE: Periodontitis belongs to a group of multifactorial diseases, characterized by inflammation and destruction of tooth-supporting tissues. P. gingivalis is one of the most important microbial factors involved in the initiation and progression of periodontitis. To survive in the host, the bacterium must acquire heme as a source of iron and protoporphyrin IX. P. gingivalis strains respond differently to changing iron and heme concentrations, which may be due to differences in the expression of systems involved in iron and heme acquisition. The ability to accumulate iron intracellularly, being different in more and less virulent P. gingivalis strains, may influence their phenotypes, production of virulence factors (including proteins engaged in heme acquisition), and virulence potential of this bacterium.

Asunto(s)

Periodontitis , Porphyromonas gingivalis , Animales , Humanos , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Hemo/metabolismo , Virulencia , Hierro/metabolismo

RESUMEN

BACKGROUND: The relationship between the major periodontal bacteria, Porphyromonas gingivalis, and the pathogenesis of IgA nephropathy (IgAN)-particularly with respect to galactose-deficient IgA1 (Gd-IgA1)-has not been fully elucidated. METHODS: Saliva samples from 30 IgAN patients and 44 patients with chronic kidney disease (CKD) were subjected to analysis of P. gingivalis status via polymerase chain reaction using a set of P. gingivalis-specific primers. The associations between P. gingivalis presence and clinical parameters, including plasma Gd-IgA1, were analyzed in each group. RESULTS: Compared with the CKD group, the IgAN group demonstrated significantly higher plasma Gd-IgA1 levels (p < 0.05). Compared with the P. gingivalis-negative subgroup, the P. gingivalis-positive subgroup exhibited significantly higher plasma Gd-IgA1 levels in both IgAN and CKD patients (p < 0.05). Additionally, among IgAN patients, the P. gingivalis-positive subgroup displayed significantly higher plasma Gd-IgA1 and urine protein levels, compared with the P. gingivalis-negative subgroup (p < 0.05). With respect to renal biopsy findings, the frequencies of segmental glomerulosclerosis and tubular atrophy/interstitial fibrosis were significantly greater in the P. gingivalis-positive subgroup than in the P. gingivalis-negative subgroup, according to the Oxford classification of IgAN (p < 0.05). CONCLUSION: Our findings suggest an association between the presence of P. gingivalis in the oral cavity and the pathogenesis of IgAN, mediated by increased levels of Gd-IgA1.

Asunto(s)

Glomerulonefritis por IGA , Insuficiencia Renal Crónica , Humanos , Glomerulonefritis por IGA/patología , Porphyromonas gingivalis/metabolismo , Galactosa/metabolismo , Inmunoglobulina A/metabolismo , Boca

RESUMEN

OBJECTIVE: This study aimed to investigate the impact of Corydalis Saxicola Bunting Total Alkaloid (CSBTA) on Porphyromonas gingivalis internalization within macrophages and explore the potential role of Toll-Like Receptor 2 (TLR2) in this process. METHODS: We established a P. gingivalis internalization model in macrophages by treating P. gingivalis-infected macrophages (MOI=100:1) with 200 µg/mL metronidazole and 300 µg/mL gentamicin for 1 h. Subsequently, the model was exposed to CSBTA at concentrations of 0.02 g/L or 1 µg/mL Pam3CSK4. After a 6 h treatment, cell lysis was performed with sterile water to quantify bacterial colonies. The mRNA expressions of TLR2 and interleukin-8 (IL-8) in macrophages were analyzed using RT-qPCR, while their protein levels were assessed via Western blot and ELISA respectively. RESULTS: P. gingivalis could internalize into macrophages and enhance the expression of TLR2 and IL-8. Activation of TLR2 by Pam3CSK4 contributed to P. gingivalis survival within macrophages and increased TLR2 and IL-8 expression. Conversely, 0.02 g/L CSBTA effectively cleared intracellular P. gingivalis, achieving a 90 % clearance rate after 6 h. Moreover, it downregulated the expression of TLR2 and IL-8 induced by P. gingivalis. However, the inhibitory effect of CSBTA on the internalized P. gingivalis model was attenuated by Pam3CSK4. CONCLUSION: CSBTA exhibited the ability to reduce the presence of live intracellular P. gingivalis and lower IL-8 expression in macrophages, possibly by modulating TLR2 activity.

Asunto(s)

Alcaloides , Corydalis , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Porphyromonas gingivalis/metabolismo , Corydalis/metabolismo , Alcaloides/metabolismo , Alcaloides/farmacología , Macrófagos/microbiología

RESUMEN

BACKGROUND: Recent epidemiological studies suggested correlation between gastric cancer (GC) and periodontal disease. AIMS: We aim to clarify involvement of lipopolysaccharide of Porphyromonas gingivalis (Pg.), one of the red complex periodontal pathogens, in the GC development. METHODS: To evaluate barrier function of background mucosa against the stimulations, we applied biopsy samples from 76 patients with GC using a Ussing chamber system (UCs). K19-Wnt1/C2mE transgenic (Gan) mice and human GC cell-lines ± THP1-derived macrophage was applied to investigate the role of Pg. lipopolysaccharide in inflammation-associated carcinogenesis. RESULTS: In the UCs, Pg. lipopolysaccharide reduced the impedance of metaplastic and inflamed mucosa with increases in mRNA expression of toll-like receptor (TLR) 2, tumor necrosis factor (TNF) α, and apoptotic markers. In vitro, Pg. lipopolysaccharide promoted reactive oxidative stress (ROS)-related apoptosis as well as activated TLR2-ß-catenin-signaling on MKN7, and it increased the TNFα production on macrophages, respectively. TNFα alone activated TLR2-ß-catenin-signaling in MKN7, while it further increased ROS and TNFα in macrophages. Under coculture with macrophages isolated after stimulation with Pg. lipopolysaccharide, ß-catenin-signaling in MKN7 was activated with an increase in supernatant TNFα concentration, both of which were decreased by adding a TNFα neutralization antibody into the supernatant. In Gan mice with 15-week oral administration of Pg. lipopolysaccharide, tumor enlargement with ß-catenin-signaling activation were observed with an increase in TNFα with macrophage infiltration. CONCLUSIONS: Local exposure of Pg. lipopolysaccharide may increase ROS on premalignant gastric mucosa to induce apoptosis-associated barrier dysfunction and to secrete TNFα from activated macrophages, and both stimulation of Pg. lipopolysaccharide and TNFα might activate TLR2-ß-catenin-signaling in GC.

Asunto(s)

Gastritis , Porphyromonas gingivalis , Humanos , Animales , Ratones , Porphyromonas gingivalis/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Lipopolisacáridos/metabolismo , beta Catenina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Membrana Mucosa/metabolismo , Carcinogénesis

RESUMEN

MicroRNA functions as an important part of the activity and development of immune cells. miR-499 has been demonstrated to play a significant role in the activity and development of immune cells. The precise mechanism by which miR-499 regulates the inflammatory response, however, remains unclear. This study was aimed to examine the role of microRNA miR-499 in the regulation of the inflammatory response in macrophages. RAW 264.7 macrophages were used as a cell model. The levels of miR-499 were measured in Porphyromonas gingivalis LPS-stimulated macrophages using qRT-PCR, and the levels of inflammatory cytokines (IL-6, IL-1ß, and TNF-α) were determined using both qRT-PCR and ELISA. StarBase was used to predict the binding sites between NRIP1 and miR-499, and the mRNA expression of NRIP1 was measured using qRT-PCR. The regulation of inflammatory factors controlled by miR-499 was also evaluated by using miR-499 inhibitor and sh-NRIP1. The activation of the JAK/STAT pathway was determined using western blotting to measure the levels of phosphorylated JAK2 and STAT1. Porphyromonas gingivalis LPS caused a high expression of miR-499, which promoted the inflammatory response in macrophages. miR-499 targeted the NRIP1 3' UTR and regulated the mRNA expression of inflammatory cytokines, including IL-6, IL-1ß, and TNF-α. The positive correlation between miR-499 and the expression of inflammatory factors and the negative correlation between NRIP1 and miR-499 suggests that the regulation of inflammatory factors controlled by miR-499 was associated with NRIP1. The phosphorylated proteins of the JAK/STAT pathway (p-JAK2 and p-STAT1) were activated by miR-499 through its regulation of NRIP1. These findings suggest that miR-499 regulates the P. gingivalis LPS-induced inflammatory response in macrophages and activates the JAK/STAT pathway through the regulation of NRIP1.

Asunto(s)

MicroARNs , Factor de Necrosis Tumoral alfa , Animales , Ratones , Citocinas/genética , Citocinas/metabolismo , Interleucina-6/metabolismo , Quinasas Janus/genética , Quinasas Janus/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/genética , Factores de Transcripción STAT/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular