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.

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.

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.

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.

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.

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.

Porphyromonas gingivalis Outer Membrane Vesicles as the Major Driver of and Explanation for Neuropathogenesis, the Cholinergic Hypothesis, Iron Dyshomeostasis, and Salivary Lactoferrin in Alzheimer's Disease.

Porphyromonas gingivalis (Pg) is a primary oral pathogen in the widespread biofilm-induced "chronic" multi-systems inflammatory disease(s) including Alzheimer's disease (AD). It is possibly the only second identified unique example of a biological extremophile in the human body. Having a better understanding of the key microbiological and genetic mechanisms of its pathogenesis and disease induction are central to its future diagnosis, treatment, and possible prevention. The published literature around the role of Pg in AD highlights the bacteria's direct role within the brain to cause disease. The available evidence, although somewhat adopted, does not fully support this as the major process. There are alternative pathogenic/virulence features associated with Pg that have been overlooked and may better explain the pathogenic processes found in the "infection hypothesis" of AD. A better explanation is offered here for the discrepancy in the relatively low amounts of "Pg bacteria" residing in the brain compared to the rather florid amounts and broad distribution of one or more of its major bacterial protein toxins. Related to this, the "Gingipains Hypothesis", AD-related iron dyshomeostasis, and the early reduced salivary lactoferrin, along with the resurrection of the Cholinergic Hypothesis may now be integrated into one working model. The current paper suggests the highly evolved and developed Type IX secretory cargo system of Pg producing outer membrane vesicles may better explain the observed diseases. Thus it is hoped this paper can provide a unifying model for the sporadic form of AD and guide the direction of research, treatment, and possible prevention.

Porphyromonas gingivalis Promotes Colorectal Carcinoma by Activating the Hematopoietic NLRP3 Inflammasome.

Porphyromonas gingivalis (P. gingivalis) is a keystone periodontal pathogen associated with various digestive cancers. However, whether P. gingivalis can promote colorectal cancer and the underlying mechanism associated with such promotion remains unclear. In this study, we found that P. gingivalis was enriched in human feces and tissue samples from patients with colorectal cancer compared with those from patients with colorectal adenoma or healthy subjects. Cohort studies demonstrated that P. gingivalis infection was associated with poor prognosis in colorectal cancer. P. gingivalis increased tumor counts and tumor volume in the ApcMin/+ mouse model and increased tumor growth in orthotopic rectal and subcutaneous carcinoma models. Furthermore, orthotopic tumors from mice exposed to P. gingivalis exhibited tumor-infiltrating myeloid cell recruitment and a proinflammatory signature. P. gingivalis promoted colorectal cancer via NLRP3 inflammasome activation in vitro and in vivo. NLRP3 chimeric mice harboring orthotopic tumors showed that the effect of NLRP3 on P. gingivalis pathogenesis was mediated by hematopoietic sources. Collectively, these data suggest that P. gingivalis contributes to colorectal cancer neoplasia progression by activating the hematopoietic NLRP3 inflammasome. SIGNIFICANCE: This study demonstrates that the periodontal pathogen P. gingivalis can promote colorectal tumorigenesis by recruiting myeloid cells and creating a proinflammatory tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2745/F1.large.jpg.

A bacterial tyrosine phosphatase modulates cell proliferation through targeting RGCC.

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

Comparing the sub-gingival levels of Cytomegalovirus, Epstein-Barr virus, Porphyromonas gingivalis in human immunodeficiency virus-1 seropositive patients with and without antiretroviral therapy.

OBJECTIVES: The effect of antiretroviral therapy (ART) on the oral pathogenic microbes in human immunodeficiency virus-1 seropositive patients remains relatively unexplored. Thus, the present study assessed the effect of ART on the sub-gingival levels of 3 pathogenic microbes. MATERIALS AND METHODS: The study groups consisted of 60 human immunodeficiency virus-1 seropositive patients divided into 3 groups of 20 each. Group 1 had periodontitis and did not start with the ART. Group 2 had periodontitis and started with ART (Tenofovir Disoproxil Fumarate 300 mg + Lamivudine 300 mg + Efavirenz 600 mg) at least 6 months before the study. Group 3 with normal periodontium, and have not started ART. The sub-gingival loads of Cytomegalovirus, Epstein-Barr virus, and the Porphyromonas gingivalis levels were assessed, along with the CD4 counts. RESULTS: The cytomegalovirus load was highest in group 1, followed by groups 2, and 3 (p-value of 0.271). The Epstein-Barr load was highest for group 2, followed by group 3, and 1 (p-value of 0.022). The P.gingivalis load was highest in group 2, followed by groups 1 and 3, (p-value of 0.028). The Epstein-Barr and Cytomegalovirus counts were significantly higher (p-value < 0.02) when the CD4 counts were less than 500 cells/cu3. CONCLUSION: ART did not cause any significant reduction in the sub-gingival levels of any of the 3 examined microbes. Given the lack of any significant effect on the sub-gingival microbial loads by the ART, human immunodeficiency virus patients may require additional anti-microbial agents and regular mechanical plaque removal to maintain their periodontal status.

Molecular Strategies Underlying Porphyromonas gingivalis Virulence.

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is considered the keystone of periodontitis diseases, a set of inflammatory conditions that affects the tissues surrounding the teeth. In the recent years, the major virulence factors exploited by P. gingivalis have been identified and characterized, including a cocktail of toxins, mainly proteases called gingipains, which promote gingival tissue invasion. These effectors use the Sec pathway to cross the inner membrane and are then recruited and transported across the outer membrane by the type IX secretion system (T9SS). In P. gingivalis, most secreted effectors are attached to anionic lipopolysaccharides (A-LPS), and hence form a virulence coat at the cell surface. P. gingivalis produces additional virulence factors to evade host immune responses, such as capsular polysaccharide, fimbriae and outer membrane vesicles. In addition to periodontitis, it is proposed that this broad repertoire of virulence factors enable P. gingivalis to be involved in diverse human diseases such as rheumatoid arthritis, and neurodegenerative, Alzheimer, and cardiovascular disorders. Here, we review the major virulence determinants of P. gingivalis and discuss future directions to better understand their mechanisms of action.

Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen responsible for severe and chronic manifestations of periodontal disease, which is one of the most common infectious disorders of humans. Although human gingival epithelium prevents intrusions by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. To study the dynamics and the fate of intracellular P. gingivalis, confocal laser scanning microscopy (CLSM) is a method of choice. Information gained with CLSM contains not only the number of P. gingivalis associated with gingival epithelial cells but also the bacterial localization on/inside the host cells, morphological change of host cells, and physical interaction between the bacteria and host organelle. In this chapter, we describe the protocols for microscopy techniques to morphologically study gingival epithelial cells infected by P. gingivalis.

Inherently and Conditionally Essential Protein Catabolism Genes of Porphyromonas gingivalis.

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.

Effects of Porphyromonas gingivalis and Its Underlying Mechanisms on Alzheimer-Like Tau Hyperphosphorylation in Sprague-Dawley Rats.

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1ß and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1ß induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1ß-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.

Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation.

Diverse microbial signatures within the intestinal microbiota have been associated with intestinal and systemic inflammatory diseases, but whether these candidate microbes actively modulate host phenotypes or passively expand within the altered microbial ecosystem is frequently not known. Here we demonstrate that colonization of mice with a member of the genus Prevotella, which has been previously associated to colitis in mice, exacerbates intestinal inflammation. Our analysis revealed that Prevotella intestinalis alters composition and function of the ecosystem resulting in a reduction of short-chain fatty acids, specifically acetate, and consequently a decrease in intestinal IL-18 levels during steady state. Supplementation of IL-18 to Prevotella-colonized mice was sufficient to reduce intestinal inflammation. Hence, we conclude that intestinal Prevotella colonization results in metabolic changes in the microbiota, which reduce IL-18 production and consequently exacerbate intestinal inflammation, and potential systemic autoimmunity.