Prevotella intermedia boosts OSCC progression through ISG15 upregulation: a new target for intervention.

PURPOSE: Periodontitis-associated bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are closely linked to the risk of oral squamous cell carcinoma (OSCC). Emerging studies have indicated that another common periodontal pathogen, Prevotella intermedia (P. intermedia), is enriched in OSCC and could affect the occurrence and progression of OSCC. Our aim is to determine the effects of P. intermedia on the progression of OSCC and the role of antibiotics in reversing these effects. METHODS: In this study, a murine xenograft model of OSCC was established, and the mice were injected intratumorally with PBS (control group), P. intermedia (P.i group), or P. intermedia combined with an antibiotic cocktail administration (P.i + ABX group), respectively. The effects of P. intermedia and ABX administration on xenograft tumor growth, invasion, angiogenesis, and metastasis were investigated by tumor volume measurement and histopathological examination. Enzyme-linked immunosorbent assay (ELISA) was used to investigate the changes in serum cytokine levels. Immunohistochemistry (IHC) was adopted to analyze the alterations in the levels of inflammatory cytokines and infiltrated immune cells in OSCC tissues of xenograft tumors. Transcriptome sequencing and analysis were conducted to determine differential expression genes among various groups. RESULTS: Compared with the control treatment, P. intermedia treatment significantly promoted tumor growth, invasion, angiogenesis, and metastasis, markedly affected the levels of inflammatory cytokines, and markedly altered M2 macrophages and regulatory T cells (Tregs) infiltration in the tumor microenvironment. However, ABX administration clearly abolished these effects of P. intermedia. Transcriptome and immunohistochemical analyses revealed that P. intermedia infection increased the expression of interferon-stimulated gene 15 (ISG15). Correlation analysis indicated that the expression level of ISG15 was positively correlated with the Ki67 expression level, microvessel density, serum concentrations and tissue expression levels of inflammatory cytokines, and quantities of infiltrated M2 macrophages and Tregs. However, it is negatively correlated with the quantities of infiltrated CD4+ and CD8+ T cells. CONCLUSION: In conclusion, intratumoral P. intermedia infection aggravated OSCC progression, which may be achieved through upregulation of ISG15. This study sheds new light on the possible pathogenic mechanism of intratumoral P. intermedia in OSCC progression, which could be a prospective target for OSCC prevention and treatment.

Association of DOK3 and infiltrated tumor-associated macrophages with risk for the prognosis of Porphyromonas gingivalis-infected oral cancer: a 12-year data analysis of 200 patients from a tertiary teaching hospital, Urumqi, China.

BACKGROUND: While there is an understanding of the association between the expression of Porphyromonas gingivalis (P. gingivalis) and prognosis of oral squamous cell carcinoma (OSCC), significance specially to address the relevance between different immunohistochemical intensities of P. gingivalis and tumor-associated macrophages (TAMs) in OSCC tissue and related clinicopathologic characteristics has not been well investigated. The present study aimed to investigate the pathological features related to M2-TAM in P. gingivalis-infected OSCC and ascertain its clinical relevance with patients' prognosis. METHODS: A prospective cohort study was designed to comparatively analyze 200 patients from June 2008 to June 2020. Bioinformatics analyses were implemented to identify DOK3 as a key molecule and to appraise immunocyte infiltration using Gene Expression Omnibus and The Cancer Genome Atlas databases. Immunohistochemical evaluation was performed to analyze the association between the expression levels of P. gingivalis, DOK3, and M2-TAM and clinicopathological variables using Fisher's exact test or Pearson's chi-square test. Cox analysis was used to calculate hazard ratios (HR) with corresponding 95% confidence interval (CI) for various clinicopathological features. The Kaplan-Meier approach and log-rank test were used to plot the survival curves. RESULTS: The expression level of P. gingivalis was positively associated with DOK3 and M2-TAMs expression level (P < 0.001). Parameters, including body mass index, clinical stage, recurrence, tumor differentiation, and P. gingivalis, DOK3, and M2-TAM immunoexpression levels, affected the prognosis of patients with OSCC (all P < 0.05). In addition, P. gingivalis (HR = 1.674, 95%CI 1.216-4.142, P = 0.012), DOK3 (HR = 1.881, 95%CI 1.433-3.457, P = 0.042), and M2-TAM (HR = 1.649, 95%CI 0.824-3.082, P = 0.034) were significantly associated with the 10-year cumulative survival rate. CONCLUSIONS: Elevated expression of P. gingivalis and DOK3 indicates M2-TAM infiltration and unfavorable prognosis of OSCC, and could be considered as three novel independent risk factors for predicting the prognosis of OSCC.

Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

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.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Refractory pneumonia caused by Prevotella heparinolytica: a case report.

BACKGROUND: Prevotella heparinolytica is a Gram-negative bacterium that is commonly found in the oral, intestinal, and urinary tracts. It has been extensively studied in lower respiratory tract infections in horses, which has heparinolytic activity and can secrete heparinase and further induces virulence factors in cells and causes disease. However, no such cases have been reported in humans. CASE PRESENTATION: A 58-year-old male patient from China presented to the respiratory clinic in Suzhou with a productive cough producing white sputum for 20 days and fever for 3 days. Prior to this visit, a chest computed tomography scan was conducted, which revealed multiple patchy nodular opacities in both lungs. On admission, the patient presented with a temperature of 38.1 °C and a pulse rate of 110 beats per minute. Despite routine anti-infective treatment with moxifloxacin, his temperature fluctuated and the treatment was ineffective. The patient was diagnosed with Prevotella heparinolytica infection through metagenomic next-generation sequencing. Therefore, the antibiotics were switched to piperacillin-tazobactam in combination with ornidazole, which alleviated his symptoms; 1 week after discharge, the patient returned to the clinic for a follow-up chest computed tomography, and the opacities on the lungs continued to be absorbed. CONCLUSION: Prevotella heparinolytica is an opportunistic pathogen. However, it has not been reported in human pneumonia. In refractory pneumonia, measures such as metagenomic next-generation sequencing can be used to identify pathogens and help guide antibiotic selection and early support.

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

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.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by stimulating the release of neutrophil extracellular traps in the tumor immune microenvironment.

BACKGROUND: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment. METHODS: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models. RESULTS: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis. CONCLUSION: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC.

Activation of liver X receptors suppresses the abundance and osteoclastogenic potential of osteoclast precursors and periodontal bone loss.

Liver-X receptors (LXRs) are essential nuclear hormone receptors involved in cholesterol and lipid metabolism. They are also believed to regulate inflammation and physiological and pathological bone turnover. We have previously shown that infection with the periodontal pathogen Porphyromonas gingivalis (Pg) in mice increases the abundance of CD11b+c-fms+Ly6Chi cells in bone marrow (BM), spleen (SPL), and peripheral blood. These cells also demonstrated enhanced osteoclastogenic activity and a distinctive gene profile following Pg infection. Here, we investigated the role of LXRs in regulating these osteoclast precursors (OCPs) and periodontal bone loss. We found that Pg infection downregulates the gene expression of LXRs, as well as ApoE, a transcription target of LXRs, in CD11b+c-fms+Ly6Chi OCPs. Activation of LXRs by treatment with GW3965, a selective LXR agonist, significantly decreased Pg-induced accumulation of CD11b+c-fms+Ly6Chi population in BM and SPL. GW3965 treatment also significantly suppressed the osteoclastogenic potential of these OCPs induced by Pg infection. Furthermore, the activation of LXRs reduces the abundance of OCPs systemically in BM and locally in the periodontium, as well as mitigates gingival c-fms expression and periodontal bone loss in a ligature-induced periodontitis model. These data implicate a novel role of LXRs in regulating OCP abundance and osteoclastogenic potential in inflammatory bone loss.

Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells.

OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Oral and Intestinal Bacterial Substances Associated with Disease Activities in Patients with Rheumatoid Arthritis: A Cross-Sectional Clinical Study.

Intestinal bacterial compositions of rheumatoid arthritis (RA) patients have been reported to be different from those of healthy people. Dysbiosis, imbalance of the microbiota, is widely known to cause gut barrier damage, resulting in an influx of bacteria and their substances into host bloodstreams in animal studies. However, few studies have investigated the effect of bacterial substances on the pathophysiology of RA. In this study, eighty-seven active RA patients who had inadequate responses to conventional synthetic disease-modifying antirheumatic drugs or severe comorbidities were analyzed for correlations between many factors such as disease activities, disease biomarkers, intestinal bacterial counts, fecal and serum lipopolysaccharide (LPS), LPS-binding protein (LBP), endotoxin neutralizing capacity (ENC), and serum antibacterial substance IgG and IgA antibody levels by multiple regression analysis with consideration for demographic factors such as age, sex, smoking, and methotrexate treatment. Serum LBP levels, fecal LPS levels, total bacteria counts, serum anti-LPS from Porphyromonas gingivalis (Pg-LPS) IgG antibody levels, and serum anti-Pg-LPS IgA antibody levels were selected for multiple regression analysis using Spearman's correlation analysis. Serum LBP levels were correlated with disease biomarker levels, such as erythrocyte sedimentation rate (p < 0.001), C-reactive protein (p < 0.001), matrix metalloproteinase-3 (p < 0.001), and IL-6 (p = 0.001), and were inversely correlated with hemoglobin (p = 0.005). Anti-Pg-LPS IgG antibody levels were inversely correlated with activity indices such as patient global assessments using visual analogue scale (VAS) (p = 0.002) and painVAS (p < 0.001). Total bacteria counts were correlated with ENC (p < 0.001), and inversely correlated with serum LPS (p < 0.001) and anti-Pg-LPS IgA antibody levels (p < 0.001). These results suggest that substances from oral and gut microbiota may influence disease activity in RA patients.

A quantitative framework reveals traditional laboratory growth is a highly accurate model of human oral infection.

Bacterial behavior and virulence during human infection is difficult to study and largely unknown, as our vast knowledge of infection microbiology is primarily derived from studies using in vitro and animal models. Here, we characterize the physiology of Porphyromonas gingivalis, a periodontal pathogen, in its native environment using 93 published metatranscriptomic datasets from periodontally healthy and diseased individuals. P. gingivalis transcripts were more abundant in samples from periodontally diseased patients but only above 0.1% relative abundance in one-third of diseased samples. During human infection, P. gingivalis highly expressed genes encoding virulence factors such as fimbriae and gingipains (proteases) and genes involved in growth and metabolism, indicating that P. gingivalis is actively growing during disease. A quantitative framework for assessing the accuracy of model systems showed that 96% of P. gingivalis genes were expressed similarly in periodontitis and in vitro midlogarithmic growth, while significantly fewer genes were expressed similarly in periodontitis and in vitro stationary phase cultures (72%) or in a murine abscess infection model (85%). This high conservation in gene expression between periodontitis and logarithmic laboratory growth is driven by overall low variance in P. gingivalis gene expression, relative to other pathogens including Pseudomonas aeruginosa and Staphylococcus aureus Together, this study presents strong evidence for the use of simple test tube growth as the gold standard model for studying P. gingivalis biology, providing biological relevance for the thousands of laboratory experiments performed with logarithmic phase P. gingivalis Furthermore, this work highlights the need to quantitatively assess the accuracy of model systems.

The role of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in chronic periodontitis progression.

Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is a novel pro-inflammatory factor in severe human diseases. Since inflammatory plays important roles in periodontitis progression, we aimed to explore the role of NEAT1 in chronic periodontitis (CP) in vitro. We established a periodontitis cell model was established by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-induced periodontal ligament cells (PDLCs). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression of NEAT1, microRNA (miR)-200c-3p, and tumor necrosis factor receptor-associated factor 6 (TRAF6). Cell viability, inflammatory factors, and protein expression of Bcl-2, Bax, and TRAF6 were analyzed by MTT, enzyme-linked immunosorbent assay, and Western blot. The target relationships among NEAT1, miR-200c-3p, and TRAF6 were predicted by the StarBase/TargetScan software, and further validated by dual-luciferase reporter assay. In this research, NEAT1 is up-regulated in CP tissues and periodontitis model group. Silencing of NEAT1 and over-expression of miR-200c-3p enhanced cell viability and repressed apoptosis in the periodontitis model group. NEAT1 targets miR-200c-3p, and miR-200c-3p further targets TRAF6. MiR-200c-3p inhibitor or over-expression of TRAF6 reversed the promoting effect of NEAT1 knockdown on cell viability, and the inhibiting effects on inflammatory cytokines and cell apoptosis. Consequently, the silencing of NEAT1 inhibits inflammation and apoptosis via targeting miR-200c-3p/TRAF6 axis, thereby contributing to alleviate the progression of CP. This finding could provide an underlying target for the treatment of CP.

Contribution of -Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview.

Periodontitis is a non-communicable chronic inflammatory disease characterized by the progressive and irreversible breakdown of the soft periodontal tissues and resorption of teeth-supporting alveolar bone. The etiology of periodontitis involves dysbiotic shifts in the diversity of microbial communities inhabiting the subgingival crevice, which is dominated by anaerobic Gram-negative bacteria, including Porphyromonas gingivalis. Indeed, P. gingivalis is a keystone pathogen with a repertoire of attributes that allow it to colonize periodontal tissues and influence the metabolism, growth rate, and virulence of other periodontal bacteria. The pathogenic potential of P. gingivalis has been traditionally analyzed using classical biochemical and molecular approaches. However, the arrival of new techniques, such as whole-genome sequencing, metagenomics, metatranscriptomics, proteomics, and metabolomics, allowed the generation of high-throughput data, offering a suitable option for bacterial analysis, allowing a deeper understanding of the pathogenic properties of P. gingivalis and its interaction with the host. In the present review, we revise the use of the different -omics technologies and techniques used to analyze bacteria and discuss their potential in studying the pathogenic potential of P. gingivalis.

Associations of Porphyromonas gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters and Survival of Esophageal Squamous Cell Carcinoma Patients.

Purpose: The present study focused on exploring the associations of Porphyromonas gingivalis (P. gingivalis) infection and low Beclin1 expression with clinicopathological parameters and survival of esophageal squamous cell carcinoma (ESCC) patients, so as to illustrate its clinical significance and prognostic value. Methods: Immunohistochemistry (IHC) was used to detect P. gingivalis infection status and Beclin1 expression in 370 ESCC patients. The chi-square test was adopted to illustrate the relationship between categorical variables, and Cohen's kappa coefficient was used for correlation analysis. Kaplan-Meier survival curves with the log-rank test were used to analyse the correlation of P. gingivalis infection and low Beclin1 expression with survival time. The effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptotic abilities of ESCC cells in vitro were detected by Cell Counting Kit-8, wound healing and flow cytometry assays. For P. gingivalis infection of ESCC cells, cell culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70-80%, cells were inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10. Result: P. gingivalis infection was negatively correlated with Beclin1 expression in ESCC tissues, and P. gingivalis infection and low Beclin1 expression were associated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis in ESCC patients. In vitro experiments confirmed that P. gingivalis infection and Beclin1 downregulation potentiate the proliferation, migration and antiapoptotic abilities of ESCC cells (KYSE150 and KYSE30). Our results provide evidence that P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC. Conclusion: Long-term smoking and alcohol consumption causes poor oral and esophageal microenvironments and ESCC patients with these features were more susceptible to P. gingivalis infection and persistent colonization, and exhibited lower Beclin1 expression, worse prognosis and more advanced clinicopathological features. Our findings indicate that effectively eliminating P. gingivalis colonization and restoring Beclin1 expression in ESCC patients may contribute to preventation and targeted treatment, and yield new insights into the aetiological research on ESCC.

Porphyromonas gingivalis Components/Secretions Synergistically Enhance Pneumonia Caused by Streptococcus pneumoniae in Mice.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis.

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV-visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

Label-free quantitative proteomic analysis of the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis to identify protein features relevant in biofilm formation.

BACKGROUND: The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. This study investigated interactions between F. nucleatum and P. gingivalis proteomes with the objective to identify proteins relevant in biofilm formation. METHODS: We applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells grown in biofilm or planktonic culture, and as mono- and dual-species models. The detected proteins were classified into functional categories and their label-free quantitative (LFQ) intensities statistically compared. RESULTS: The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins, including abundant virulence factors. Using univariate statistics, we identified significant changes between biofilm and planktonic culture (p-value ≤0.05) in 0,4% F. nucleatum, 7% P. gingivalis, and 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolism had significantly increased levels in biofilm. In both mono- and dual-species biofilms, P. gingivalis increased the production of proteins for translation, oxidation-reduction, and amino acid metabolism compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. CONCLUSIONS: The findings suggest that P. gingivalis reduces the production of multiple proteins because of the F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilms, which need to be considered in the design of prevention strategies.

The effects of periodontitis associated microbiota on the development of oral squamous cell carcinoma.

Epidemiological data have shown that periodontal bacterial infection, periodontitis, and oral squamous cell carcinoma have close relationship on the disease progress and risk. However, the specific role of periodontal microbes and their mechanism in the development of oral squamous cell carcinoma is not yet clear. In our previous work, metagenomic Illumina Mi-seq analysis was used to identify tstructure and abundance of periodontital microbiome. Accoding to the results, we used Porphyromonas.spp. and Fusobacterium.spp. as the periodontitis positive microbiota; Neisseria.spp and Corynebacterium.spp as periodontitis negative microbiota (their average relative abundance were >5%). These representative strains of the above genus were used to infect OSCC cells to explore their effect on tumor cell biology behavior, and detect the expression level of the gene in related to inflammation, migration, invasion and cell cycle. We find that periodontitis positive correlated microbiota had a promoting effect on the development of oral squamous cell carcinoma in vitro by regulating mRNA and protein expression of IL-6, IL-8, MMP-9 and Cyclin-D1. Periodontitis negative correlated microbiota had suppression effect on the development of oral squamous cell carcinoma in vitro analysis.

Probiotics improve re-epithelialization of scratches infected by Porphyromonas gingivalis through up-regulating CXCL8-CXCR1/CXCR2 axis.

Porphyromonas gingivalis inhibits the release of CXCL8 by gingival epithelial cells and reduces their proliferation. We previously reported that Bifidocaterium sp. and Lactobacillus sp. immunomodulate gingival epithelial cells response to this periodontal pathogen, but their effects on re-epithelialization properties are still unknown. Herein we explored these activities of potential probiotics on gingival epithelial cells and clarified their mechanisms. The immortalized OBA-9 lineage was used to perform in vitro scratches. Twelve clinical isolates and commercially available strains of Bifidobacterium sp. and Lactobacillus sp. were screened. L. casei 324 m and B. pseudolongum 1191A were selected to perform mechanistic assays with P. gingivalis W83 infection and the following parameters were measured: percentage of re-epithelialization by DAPI immunofluorescence area measurement; cell number by Trypan Blue exclusion assay; CXCL8 regulation by ELISA and RT-qPCR; and expression of CXCL8 cognate receptors-CXCR1 and CXCR2 by Flow Cytometry. Complementary mechanistic assays were performed with CXCL8, in the presence or absence of the CXCR1/CXCR2 inhibitor-reparixin. L. casei 324 m and B. pseudolongum 1191A enhanced re-epithelialization/cell proliferation as well as inhibited the harmful effects of P. gingivalis W83 on these activities through an increase in the expression and release of CXCL8 and in the number of cells positive for CXCR1/CXCR2. Further, we revealed that the beneficial effects of these potential probiotics were dependent on activation of the CXCL8-CXCR1/CXCR2 axis. The current findings indicate that these potential probiotics strains may improve wound healing in the context of the periodontal tissues by a CXCL8 dependent mechanism.