ABSTRACT
GV1001, an anticancer vaccine, exhibits other biological functions, including anti-inflammatory and antioxidant activity. It also suppresses the development of ligature-induced periodontitis in mice. Porphyromonas gingivalis (Pg), a major human oral bacterium implicated in the development of periodontitis, is associated with various systemic disorders, such as atherosclerosis and Alzheimer's disease (AD). This study aimed to explore the protective effects of GV1001 against Pg-induced periodontal disease, atherosclerosis, and AD-like conditions in Apolipoprotein (ApoE)-deficient mice. GV1001 effectively mitigated the development of Pg-induced periodontal disease, atherosclerosis, and AD-like conditions by counteracting Pg-induced local and systemic inflammation, partly by inhibiting the accumulation of Pg DNA aggregates, Pg lipopolysaccharides (LPS), and gingipains in the gingival tissue, arterial wall, and brain. GV1001 attenuated the development of atherosclerosis by inhibiting vascular inflammation, lipid deposition in the arterial wall, endothelial to mesenchymal cell transition (EndMT), the expression of Cluster of Differentiation 47 (CD47) from arterial smooth muscle cells, and the formation of foam cells in mice with Pg-induced periodontal disease. GV1001 also suppressed the accumulation of AD biomarkers in the brains of mice with periodontal disease. Overall, these findings suggest that GV1001 holds promise as a preventive agent in the development of atherosclerosis and AD-like conditions associated with periodontal disease.
Subject(s)
Apolipoproteins E , Atherosclerosis , Periodontal Diseases , Porphyromonas gingivalis , Animals , Mice , Apolipoproteins E/deficiency , Periodontal Diseases/microbiology , Periodontal Diseases/prevention & control , Atherosclerosis/metabolism , Atherosclerosis/prevention & control , Atherosclerosis/microbiology , Telomerase/metabolism , Peptide Fragments/pharmacology , Alzheimer Disease/metabolism , Alzheimer Disease/prevention & control , Alzheimer Disease/microbiology , Periodontitis/microbiology , Periodontitis/prevention & control , Bacteroidaceae Infections/microbiology , Bacteroidaceae Infections/complications , Bacteroidaceae Infections/prevention & control , Disease Models, Animal , Mice, Inbred C57BL , Male , HumansABSTRACT
Porphyromonas gingivalis is a Gram-negative pathogenic bacterium associated with chronic periodontitis. The development of a chimeric peptide-based vaccine targeting this pathogen could be highly beneficial in preventing oral bone loss as well as other severe gum diseases. We applied a computational framework to design a multi-epitope-based vaccine candidate against P. gingivalis. The vaccine comprises epitopes from subunit proteins prioritized from the P. gingivalis reference strain (P. gingivalis ATCC 33277) using several reported vaccine properties. Protein-based subunit vaccines were prioritized through genomics techniques. Epitope prediction was performed using immunoinformatic servers and tools. Molecular modeling approaches were used to build a putative three-dimensional structure of the vaccine to understand its interactions with host immune cells through biophysical techniques such as molecular docking simulation studies and binding free energy methods. Genome subtraction identified 18 vaccine targets: six outer-membrane, nine cytoplasmic membrane-, one periplasmic, and two extracellular proteins. These proteins passed different vaccine checks required for the successful development of a vaccine candidate. The shortlisted proteins were subjected to immunoinformatic analysis to map B-cell derived T-cell epitopes, and antigenic, water-soluble, non-toxic, and good binders of DRB1*0101 were selected. The epitopes were then modeled into a multi-epitope peptide vaccine construct (linked epitopes plus adjuvant) to enhance immunogenicity and effectively engage both innate and adaptive immunity. Further, the molecular docking approach was used to determine the binding conformation of the vaccine to TLR2 innate immune receptor. Molecular dynamics simulations and binding free energy calculations of the vaccine-TLR2 complex were performed to highlight key intermolecular binding energies. Findings of this study will be useful for vaccine developers to design an effective vaccine for chronic periodontitis pathogens, specifically P. gingivalis.
Subject(s)
Bacterial Vaccines , Bacteroidaceae Infections , Porphyromonas gingivalis , Bacteroidaceae Infections/prevention & control , Chronic Periodontitis/prevention & control , Computational Biology , Epitopes, T-Lymphocyte , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Porphyromonas gingivalis/immunology , Toll-Like Receptor 2 , Vaccines, SubunitABSTRACT
Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent Porphyromonas gingivalis, and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. Juncus effusus L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from J. effusus L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from J. effusus L. significantly reduced P. gingivalis or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from J. effusus L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from P. gingivalis LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from J. effusus L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.
Subject(s)
Anti-Inflammatory Agents , Keratinocytes/metabolism , Magnoliopsida/chemistry , Mouth Mucosa/metabolism , Plant Extracts , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Bacteroidaceae Infections/metabolism , Bacteroidaceae Infections/prevention & control , Cell Line, Transformed , Humans , Keratinocytes/pathology , Mouth Mucosa/pathology , Periodontitis/metabolism , Periodontitis/pathology , Periodontitis/prevention & control , Plant Extracts/chemistry , Plant Extracts/pharmacology , Porphyromonas gingivalis/metabolismABSTRACT
It has been reported that GroEL, a heat shock protein (HSP) produced by the representative periodontopathogenic bacterium, Porphyromonas gingivalis, induces inflammation-induced osteoclastogenesis and promotes alveolar bone resorption. In this study, we demonstrated the efficacy of a mucosal vaccine targeting GroEL against bone resorption induced by P. gingivalis. Female BALB/c mice received sublingual CpG oligodeoxynucleotide as an adjuvant with recombinant GroEL (rGroEL) prior to P. gingivalis exposure. Animals were euthanized 30 days after P. gingivalis inoculation. Sublingual immunization (SLI) with rGroEL elicited significant rGroEL-specific serum immunoglobulin (Ig)G and salivary IgA antibody (Ab) responses, and these responses were sustained for approximately 1 year. Interestingly, 10-fold more GroEL-specific IgA Ab-producing cells were detected in the submandibular glands (SMGs) than in the spleen. Antigen (Ag)-specific cells isolated from the spleen and SMGs induced significantly higher levels of IFN-γ expression after Ag restimulation in vitro. Flow cytometry illustrated that the frequency of CD11b+ dendritic cells with enhanced expression of CD80, CD86, CD40, and major histocompatibility complex II molecules was significantly increased in the SMGs. Furthermore, SLI with rGroEL significantly suppressed P. gingivalis-induced alveolar bone resorption and P. gingivalis-stimulated tumor necrosis factor-α, interleukin-6, and HSP60 expression in the gingiva. These findings suggest that SLI with rGroEL and CpG oligodeoxynucleotide is a beneficial strategy for preventing periodontal disease, mainly by presenting Ags in the oral region and inducing antibody production in the mucosal and systemic systems.
Subject(s)
Alveolar Bone Loss , Bacteroidaceae Infections , Alveolar Bone Loss/microbiology , Alveolar Bone Loss/prevention & control , Animals , Antibodies, Bacterial , Bacteroidaceae Infections/prevention & control , Female , Immunization , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G , Inflammation , Mice , Mice, Inbred BALB C , Oligodeoxyribonucleotides/metabolism , Porphyromonas gingivalis/metabolismABSTRACT
Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antibodies, Bacterial/metabolism , Bacterial Vaccines/administration & dosage , Bacteroidaceae Infections/prevention & control , Fimbriae Proteins/administration & dosage , Immunogenicity, Vaccine , Immunoglobulin A, Secretory/metabolism , Porphyromonas gingivalis/immunology , Respiratory System/drug effects , Administration, Intranasal , Animals , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/microbiology , Disease Models, Animal , Female , Fimbriae Proteins/genetics , Fimbriae Proteins/immunology , Immunity, Mucosal/drug effects , Immunization Schedule , Membrane Proteins/administration & dosage , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice, Inbred C57BL , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/immunology , Porphyromonas gingivalis/pathogenicity , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Respiratory System/immunology , Respiratory System/metabolism , Respiratory System/microbiology , Time Factors , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunologyABSTRACT
Bacteria release spherical nanobodies, known as membrane vesicles (MVs), during various growth phases. MVs have been gaining recognition as structurally stable vehicles in the last two decades because they deliver a wide range of antigens, virulence factors, and immunomodulators to the host. These functions suggest not only the possible contribution of MVs to pathogenicity but also the potential applicability of low-dose MVs for use as vaccines. Here, we describe a series of methods for isolating MVs of Porphyromonas gingivalis, which is an important species among periodontopathic bacteria. The present chapter also introduces a mouse model of intranasal immunization using MVs from P. gingivalis.
Subject(s)
Bacterial Outer Membrane/immunology , Bacterial Vaccines/therapeutic use , Bacteroidaceae Infections/prevention & control , Porphyromonas gingivalis/immunology , Administration, Intranasal , Animals , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/immunology , Bacteroidaceae Infections/immunology , Centrifugation, Density Gradient/methods , Disease Models, Animal , Female , Immunization , Mice , Mice, Inbred BALB C , Ultracentrifugation/methodsABSTRACT
BACKGROUND: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). METHODS: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0-24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. RESULTS: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. CONCLUSIONS: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteroidaceae Infections/prevention & control , Chlorogenic Acid/pharmacology , Coffea/chemistry , Periodontitis/drug therapy , Plant Extracts/pharmacology , Porphyromonas gingivalis/drug effects , Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/metabolism , Bacteroidaceae Infections/microbiology , Chlorogenic Acid/isolation & purification , Disk Diffusion Antimicrobial Tests , Microbial Viability/drug effects , Peptide Hydrolases/metabolism , Periodontitis/microbiology , Plant Extracts/isolation & purification , Porphyromonas gingivalis/enzymology , Porphyromonas gingivalis/pathogenicity , Seeds/chemistry , Time Factors , Virulence Factors/metabolismABSTRACT
Pathogens are capable of hijacking immune defense mechanisms, thereby creating a tolerogenic environment for hypermutated malignant cells that arise within the site of infection. Immune checkpoint-oriented immunotherapies have shown considerable promise. Equally important, the epigenetic reprogramming of an immune-evasive phenotype that activates the immune system in a synergistic manner can improve immunotherapy outcomes. These advances have led to combinations of epigenetic- and immune-based therapeutics. We previously demonstrated that Porphyromonas gingivalis isolated from esophageal squamous cell carcinoma (ESCC) lesions represents a major pathogen associated with this deadly disease. In this study, we examined the mechanisms associated with host immunity during P. gingivalis infection and demonstrated that experimentally infected ESCC responds by increasing the expression of B7-H4 and lysine demethylase 5B, which allowed subsequent in vivo analysis of the immunotherapeutic effects of anti-B7-H4 and histone demethylase inhibitors in models of chronic infection and immunity against xenografted human tumors. Using three different preclinical mouse models receiving combined therapy, we showed that mice mounted strong resistance against P. gingivalis infection and tumor challenge. This may have occurred via generation of a T cell-mediated response in the microenvironment and formation of immune memory. In ESCC subjects, coexpression of B7-H4 and KDM5B correlated more significantly with bacterial load than with the expression of either molecule alone. These results highlight the unique ability of P. gingivalis to evade immunity and define potential targets that can be exploited therapeutically to improve the control of P. gingivalis infection and the development of associated neoplasia.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Bacteroidaceae Infections/prevention & control , Esophageal Squamous Cell Carcinoma/immunology , Immunity/drug effects , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Porphyromonas/immunology , Repressor Proteins/antagonists & inhibitors , V-Set Domain-Containing T-Cell Activation Inhibitor 1/antagonists & inhibitors , Animals , Bacteroidaceae Infections/immunology , Cell Line, Tumor , Disease Models, Animal , Epigenesis, Genetic , Esophageal Squamous Cell Carcinoma/metabolism , Gene Expression Profiling , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Immunohistochemistry , Immunophenotyping , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Mice, Transgenic , Models, Biological , Porphyromonas/genetics , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
BACKGROUND: Porphyromonas gingivalis adherence to oral streptococci is a key point in the pathogenesis of periodontal diseases (Honda in Cell Host Microbe 10:423-425, 2011). Previous work in our groups has shown that a region of the streptococcal antigen denoted BAR (SspB Adherence Region) inhibits P. gingivalis/S. gordonii interaction and biofilm formation both in vitro and in a mouse model of periodontitis (Daep et al. in Infect Immun 74:5756-5762, 2006; Daep et al. in Infect immun 76:3273-3280, 2008; Daep et al. in Infect Immun 79:67-74, 2011). However, high localized concentration and prolonged exposure are needed for BAR to be an effective therapeutic in the oral cavity. METHODS: To address these challenges, we fabricated poly(lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol PLGA (mPEG-PLGA) nanoparticles (NPs) that encapsulate BAR peptide, and assessed the potency of BAR-encapsulated NPs to inhibit and disrupt in vitro two-species biofilms. In addition, the kinetics of BAR-encapsulated NPs were compared after different durations of exposure in a two-species biofilm model, against previously evaluated BAR-modified NPs and free BAR. RESULTS: BAR-encapsulated PLGA and mPEG-PLGA NPs potently inhibited biofilm formation (IC50 = 0.7 µM) and also disrupted established biofilms (IC50 = 1.3 µM) in a dose-dependent manner. In addition, BAR released during the first 2 h of administration potently inhibits biofilm formation, while a longer duration of 3 h is required to disrupt pre-existing biofilms. CONCLUSIONS: These results suggest that BAR-encapsulated NPs provide a potent platform to inhibit (prevent) and disrupt (treat) P. gingivalis/S. gordonii biofilms, relative to free BAR.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antigens, Bacterial/pharmacology , Biofilms/drug effects , Drug Carriers/chemistry , Nanoparticles/chemistry , Porphyromonas gingivalis/drug effects , Streptococcus gordonii/drug effects , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/immunology , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/immunology , Bacterial Adhesion/drug effects , Bacteroidaceae Infections/prevention & control , Humans , Lactic Acid/chemistry , Polyethylene Glycols/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Porphyromonas gingivalis/physiology , Streptococcal Infections/prevention & control , Streptococcus/immunology , Streptococcus gordonii/physiologyABSTRACT
In previous research, to combine the immunogenicity of Fusobacterium nucleatum (F. nucleatum) and the probiotic properties of Lactobacillus acidophilus (L. acidophilus), we constructed a FomA-expressing L. acidophilus strain and assessed its immunogenicity. Our findings indicated that oral administration of the recombinant L. acidophilus strain reduced the risk of periodontal infection by Porphyromonas gingivalis (P. gingivalis) and F. nucleatum. However, because the exogenous FomA is an heterologous protein for the original bacterium, in this study, we assessed whether the biochemical characteristics of the recombinant L. acidophilus strain change due to the expression of the exogenous FomA protein. OBJECTIVES: To test the biochemical characteristics of a recombinant L. acidophilus strain expressing exogenous FomA and assess its antibiotic sensitivity. DESIGNS: We assessed the colony morphology, growth, acid production, and carbohydrate fermentation abilities of the recombinant L. acidophilus strain. In addition, we tested the adhesive ability and antimicrobial activity of the recombinant and assessed its antibiotic sensitivity through a drug susceptibility test. RESULTS: The experimental results showed that the colony and microscopic morphology of the recombinant L. acidophilus strain was consistent with the original strain, and the recombinant strain grew well when cultured under aerobic or anaerobic conditions, exhibiting a growth rate that was identical to that of the standard strain. Similarly, the supernatants of the recombinant L. acidophilus can inhibit the growth of E. coli and P. gingivalis at different concentrations, and the recombinant strain displayed essentially the same drug sensitivity profile as the original L. acidophilus. However, to our surprise, the recombinant strains exhibited a greater adhesion ability than the reference strain. CONCLUSIONS: Our study demonstrated that, in addition to an increased adhesion ability, the recombinant L. acidophilus strain maintained the basic characteristics of the standard strain ATCC 4356, including antibiotic sensitivity. Thus, the recombinant strains have great potential to be utilized as a safe and effective periodontitis vaccine in the future.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacteroidaceae Infections/therapy , Fusobacterium Infections/therapy , Lactobacillus acidophilus/metabolism , Animals , Bacterial Adhesion/drug effects , Bacterial Adhesion/immunology , Bacterial Outer Membrane Proteins/drug effects , Bacterial Outer Membrane Proteins/metabolism , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/prevention & control , Cells, Cultured , Erythromycin/pharmacology , Fusobacterium Infections/immunology , Fusobacterium Infections/prevention & control , Fusobacterium nucleatum/immunology , Fusobacterium nucleatum/pathogenicity , Glycolysis , Lactobacillus acidophilus/drug effects , Lactobacillus acidophilus/immunology , Microbial Sensitivity Tests , Porphyromonas gingivalis/immunology , Porphyromonas gingivalis/pathogenicity , Probiotics/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
The development of antibiotics cannot keep up with the speed of resistance acquired by microorganisms. Recently, the development of antimicrobial photodynamic therapy (aPDT) has been a necessary antimicrobial strategy against antibiotic resistance. Among the wide variety of bacteria found in the oral flora, Porphyromonas gingivalis (P. gingivalis) is one of the etiological agents of periodontal disease. aPDT has been studied for periodontal disease, but has risks of cytotoxicity to normal stained tissue. In this study, we performed aPDT using protoporphyrin IX (PpIX), an intracellular pigment of P. gingivalis, without an external photosensitizer. We confirmed singlet oxygen generation by PpIX in a blue-light irradiation intensity-dependent manner. We discovered that blue-light irradiation on P. gingivalis is potentially bactericidal. The sterilization mechanism seems to be oxidative DNA damage in bacterial cells. Although it is said that no resistant bacteria will emerge using aPDT, the conventional method relies on an added photosensitizer dye. PpIX in P. gingivalis is used in energy production, so aPDT applied to PpIX of P. gingivalis should limit the appearance of resistant bacteria. This approach not only has potential as an effective treatment for new periodontal diseases, but also offers potential antibacterial treatment for multiple drug resistant bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteroidaceae Infections/prevention & control , Light , Periodontal Diseases/prevention & control , Photosensitizing Agents/pharmacology , Porphyromonas gingivalis/drug effects , Singlet Oxygen/chemistry , Bacteroidaceae Infections/metabolism , Bacteroidaceae Infections/microbiology , Humans , Microbial Viability , Periodontal Diseases/microbiology , Porphyromonas gingivalis/isolation & purification , Porphyromonas gingivalis/radiation effects , Protoporphyrins/metabolismABSTRACT
Chronic periodontitis is caused by interactions between the oral polymicrobial community and host factors. Periodontal diseases are associated with dysbiotic shift in oral microbiota. Vaccination against periodontopathic bacteria could be a fundamental therapeutic to modulate polymicrobial biofilms. Because oral cavity is the site of periodontopathic bacterial colonization, mucosal vaccines should provide better protection than vaccines administered systemically. We previously reported that bacterial flagellin is an excellent mucosal adjuvant. In this study, we investigated whether mucosal immunization with a flagellin-adjuvanted polypeptide vaccine induces protective immune responses using a Porphyromonas gingivalis infection model. We used the Hgp44 domain polypeptide of Arg-gingipain A (RgpA) as a mucosal antigen. Intranasal (IN) immunization induced a significantly higher Hgp44-specific IgG titer in the serum of mice than sublingual (SL) administration. The co-administration of flagellin potentiated serum IgG responses for both the IN and SL vaccinations. On the other hand, the anti-Hgp44-specific IgA titer in the saliva was comparable between IN and SL vaccinations, suggesting SL administration as more compliant vaccination route for periodontal vaccines. The co-administration of flagellin significantly potentiated the secretory IgA response in saliva also. Furthermore, mice administered a mixture of Hgp44 and flagellin via the IN and SL routes exhibited significant reductions in alveolar bone loss induced by live P. gingivalis infections. An intranasally administered Hgp44-flagellin fusion protein induced a comparable level of Hgp44-specific antibody responses to the mixture of Hgp44 and flagellin. Overall, a flagellin-adjuvanted Hgp44 antigen would serve an important component for a multivalent mucosal vaccine against polymicrobial periodontitis.
Subject(s)
Adhesins, Bacterial/immunology , Adjuvants, Immunologic/administration & dosage , Bacterial Vaccines/immunology , Bacteroidaceae Infections/prevention & control , Cysteine Endopeptidases/immunology , Flagellin/administration & dosage , Periodontal Diseases/prevention & control , Porphyromonas gingivalis/immunology , Administration, Mucosal , Animals , Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Bacteroidaceae Infections/complications , Disease Models, Animal , Female , Gingipain Cysteine Endopeptidases , Immunoglobulin A/analysis , Immunoglobulin G/blood , Mice, Inbred BALB C , Periodontal Diseases/pathology , Saliva/immunology , Treatment Outcome , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunologyABSTRACT
Probiotics have been used to treat gastrointestinal disorders. However, the effect of orally intubated probiotics on oral disease remains unclear. We assessed the potential of oral administration of Lactobacillus gasseri SBT2055 (LG2055) for Porphyromonas gingivalis infection. LG2055 treatment significantly reduced alveolar bone loss, detachment and disorganization of the periodontal ligament, and bacterial colonization by subsequent P. gingivalis challenge. Furthermore, the expression and secretion of TNF-α and IL-6 in gingival tissue was significantly decreased in LG2055-administered mice after bacterial infection. Conversely, mouse ß-defensin-14 (mBD-14) mRNA and its peptide products were significantly increased in distant mucosal components as well as the intestinal tract to which LG2055 was introduced. Moreover, IL-1ß and TNF-α production from THP-1 monocytes stimulated with P. gingivalis antigen was significantly reduced by the addition of human ß-defensin-3. These results suggest that gastrically administered LG2055 can enhance immunoregulation followed by periodontitis prevention in oral mucosa via the gut immune system; i.e., the possibility of homing in innate immunity.
Subject(s)
Antibiosis , Bacteroidaceae Infections/microbiology , Bacteroidaceae Infections/prevention & control , Lactobacillus gasseri/physiology , Periodontal Diseases/microbiology , Periodontal Diseases/prevention & control , Porphyromonas gingivalis , Probiotics/administration & dosage , Alveolar Bone Loss/microbiology , Alveolar Bone Loss/pathology , Alveolar Bone Loss/prevention & control , Animals , Biopsy , Cytokines/metabolism , Disease Models, Animal , Inflammation Mediators/metabolism , Mice , Monocytes/metabolism , Periodontal Ligament/microbiology , Periodontal Ligament/pathology , beta-DefensinsABSTRACT
Various surface modifications have been tried for enhancing osseointegration of the dental implants like mechanical and/or chemical treatments and deposition of calcium phosphate coatings. The objective of this research was to develop calcium-phosphate based thin coatings with antibacterial and bioactive properties for potential application in dental implants. Titanium (Ti) discs were immersed in different calcifying solutions: CaP (positive control), F-CaP, Zn-CaP, and FZn-CaP and incubated for 24 h. Negative control was uncoated Ti discs. Coated surfaces were characterized using X-ray diffraction, scanning electron microscopy and energy dispersive spectroscopy. Antibacterial properties were tested using Porphyromonas gingivalis because of its strong association with periodontal and peri-implant infections. Bacterial adhesion and colonization were studied at different timepoints. The coated surfaces had compositional characteristics similar to that of bone mineral and they inhibited the growth, colonization and adherence of P. gingivalis, resulted in reduced thickness of biofilms and bacterial inhibition in the culture medium as compared to the positive and negative controls (p < 0.05). There was no significant difference between the experimental groups (p > 0.05). It has been previously demonstrated that these coatings have excellent in vitro bioactivity (formed carbonate hydroxyapatite when immersed in a simulated body fluid). Such coatings can enhance osseointegration and prevent infection in implants, thereby improving the success rates of implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2218-2227, 2017.
Subject(s)
Anti-Bacterial Agents/chemistry , Calcium Phosphates/chemistry , Coated Materials, Biocompatible/chemistry , Dental Implants/microbiology , Titanium/chemistry , Anti-Bacterial Agents/pharmacology , Bacteroidaceae Infections/prevention & control , Biofilms/drug effects , Calcium Phosphates/pharmacology , Coated Materials, Biocompatible/pharmacology , Durapatite/chemistry , Humans , Porphyromonas gingivalis/drug effects , Porphyromonas gingivalis/physiology , Surface Properties , Titanium/pharmacology , X-Ray DiffractionABSTRACT
OBJECTIVE: Following Porphyromonas gingivalis infection in mice, the efficacy of vaccination by recombinant and native RgpA in modulating the early local anti-inflammatory and immune responses and periodontal bone loss were examined. MATERIAL AND METHODS: Using the subcutaneous chamber model, exudates were analyzed for cytokines after treatment with native RgpA and adjuvant (test), or adjuvant and saline alone (controls). Mice were also immunized with recombinant RgpA after being orally infected with P. gingivalis. After 6 wk, serum was examined for anti-P. gingivalis IgG1 and IgG2a titers and for alveolar bone resorption. RESULTS: Immunization with native RgpA shifted the immune response toward an anti-inflammatory response as demonstrated by decreased proinflammatory cytokine IL-1ß production and greater anti-inflammatory cytokine IL-4 in chamber exudates. Systemically, immunization with recombinant RgpA peptide prevented alveolar bone loss by 50%, similar to immunization with heat-killed whole bacteria. Furthermore, recombinant RgpA shifted the humoral response toward high IgG1 and low IgG2a titers, representing an in vivo anti-inflammatory response. CONCLUSIONS: The present study demonstrates the potential of RgpA to shift the early local immune response toward an anti-inflammatory response while vaccination with recRgpA protected against P. gingivalis-induced periodontitis.
Subject(s)
Adhesins, Bacterial/immunology , Alveolar Bone Loss/prevention & control , Bacterial Vaccines/therapeutic use , Bacteroidaceae Infections/prevention & control , Cysteine Endopeptidases/immunology , Porphyromonas gingivalis , Alveolar Bone Loss/microbiology , Animals , Antibodies, Bacterial/immunology , Bacterial Vaccines/immunology , Bacteroidaceae Infections/immunology , Female , Gingipain Cysteine Endopeptidases , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Porphyromonas gingivalis/immunology , Vaccines, Synthetic/immunology , Vaccines, Synthetic/therapeutic useABSTRACT
Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (10(8) cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 µg/ml plantibody. Anti-FimA plantibody (10 µg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease.
Subject(s)
Fimbriae Proteins/immunology , Plantibodies/immunology , Adolescent , Adult , Animals , Bacterial Adhesion/drug effects , Bacterial Load , Bacteroidaceae Infections/microbiology , Bacteroidaceae Infections/prevention & control , Cells, Cultured , Disease Models, Animal , Endocytosis/drug effects , Female , Humans , Macrophages/immunology , Male , Mice, Inbred C57BL , Microbial Viability/drug effects , Oryza , Phagocytosis , Porphyromonas gingivalis/immunology , Porphyromonas gingivalis/physiology , Young AdultABSTRACT
Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1ß, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases.
Subject(s)
Alveolar Bone Loss/prevention & control , Bone Density Conservation Agents/therapeutic use , Osteoclasts/drug effects , Porphyromonas gingivalis/drug effects , Sodium Fluoride/therapeutic use , Acid Phosphatase/drug effects , Alveolar Bone Loss/microbiology , Animals , Anti-Bacterial Agents/therapeutic use , Anti-Inflammatory Agents/therapeutic use , Bacteroidaceae Infections/microbiology , Bacteroidaceae Infections/prevention & control , Cathepsin K/drug effects , Interleukin-1beta/drug effects , Interleukin-6/analysis , Interleukin-8/drug effects , Isoenzymes/drug effects , Macrophage Colony-Stimulating Factor/drug effects , Male , Matrix Metalloproteinase 9/drug effects , Periodontitis/microbiology , Periodontitis/prevention & control , RANK Ligand/drug effects , Rats , Rats, Sprague-Dawley , Tartrate-Resistant Acid Phosphatase , Transcription Factors/drug effects , X-Ray Microtomography/methodsABSTRACT
The outer membrane protein RagB is one of the major virulence factors of Porphyromonas gingivalis (P. gingivalis). To prevent periodontitis and associated systemic diseases induced by P. gingivalis, we built B cell antigen epitope vaccine characterized by pIRES-ragB'-mGITRL to induce a protective immune responses. The B cell antigen epitope and scrambled peptide of ragB were predicted, cloned into pIRES and constructed pIRES-ragB', pIRES-scrambled epitopes and pIRES-ragB'-mGITRL. pIRES-ragB'-mGITRL was transfected into COS-7 cells. Subsequently, the 6-week-old female BALB/c mice were challenged by P. gingivalis following three time immunization by pIRES, pIRES-ragB', pIRES-scrambled epitopes and pIRES-ragB'-mGITRL. The levels of RagB-specific antibody in the serum and Tfh cells in the spleen were measured by ELISA and flow cytometry, respectively. And higher levels of RagB-specific IgG were produced in the immunized mice with pIRES-ragB'-mGITRL. Additionally, the number of Tfh cells was also expanded and lesions were diminished in pIRES-ragB'-mGITRL mice comparing with control groups. Our results clearly demonstrated that P. gingivalis B cell antigen epitope vaccine, pIRES-ragB'-mGITRL, could induce protective immune responses. Furthermore, our data also indicated that pIRES-ragB'-mGITRL was a potential therapeutic vaccine against P. gingivalis.
Subject(s)
Antibodies, Bacterial/immunology , Bacterial Proteins/immunology , Epitopes, B-Lymphocyte/immunology , Porphyromonas gingivalis/immunology , Tumor Necrosis Factors/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Bacterial/blood , Bacterial Proteins/genetics , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/prevention & control , Bacteroidaceae Infections/virology , COS Cells , Cell Proliferation , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Immunization , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice, Inbred BALB C , Periodontitis/immunology , Periodontitis/prevention & control , Periodontitis/virology , Porphyromonas gingivalis/genetics , Porphyromonas gingivalis/physiology , Receptors, Antigen, B-Cell/immunology , Spleen/immunology , T-Lymphocytes, Helper-Inducer/immunology , Tumor Necrosis Factors/genetics , Vaccines, DNA/pharmacologyABSTRACT
The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.
Subject(s)
Adhesins, Bacterial/chemistry , Bacteroidaceae Infections/microbiology , Cysteine Endopeptidases/chemistry , Porphyromonas gingivalis/chemistry , Adhesins, Bacterial/metabolism , Bacteroidaceae Infections/prevention & control , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Gingipain Cysteine Endopeptidases , Humans , Models, Molecular , Oral Health , Porphyromonas gingivalis/metabolism , Protein ConformationABSTRACT
PURPOSE: The aim of this study was to evaluate the capability of chemicals to decontaminate Escherichia coli (E coli) or Porphyromonas gingivalis (P gingivalis) from sandblasted acid-etched (SAE) titanium dental implants. MATERIALS AND METHODS: SAE titanium dental implants were contaminated with E coli or P gingivalis and incubated in a sterile bacterial culture media under aerobic and anaerobic conditions, respectively. The implants were treated with 10 different conditions: calcium hydroxide [Ca(OH)2] paste for 1 minute and saline irrigation for 1 minute; Ca(OH)2 paste for 1 minute and 0.2% chlorhexidine digluconate (CHXD) irrigation for 1 minute; 0.2% CHXD for 1 minute; Dakin's solution for 1 minute; tetracycline hydrochloride (T-HCl) as a 1 g per 20 mL solution for 1, 2, and 3 minutes; and T-HCl paste for 1, 2, and 3 minutes. All implants were irrigated with 1 mL of saline solution and incubated under aerobic and anaerobic conditions for 24 hours or 48 hours for E coli- and P gingivalis-contaminated implants, respectively. The control group was submitted to all procedures except for the chemical treatments. Aliquots were removed, and turbidity was measured by spectrophotometry. The level of bacterial growth in control cultures was considered to have a decontamination percentage (DC%) of 0. RESULTS: Spectrophotometric analysis showed that all chemical treatments resulted in significantly higher DC% compared to controls for SAE implants contaminated with E coli (P < .05) or P gingivalis (P < .05). For the P gingivalis experiments, SAE implants treated with Ca(OH)2 paste and saline solution had a lower DC% (39.3%) than those in the other treatment groups. In the E coli experiments, DC% was significantly lower for SAE implants treated with Ca(OH)2 paste and saline solution (48.7%), Dakin's solution (92.7%), or T-HCl paste for 1 minute (96.6%) than those in the other groups. CONCLUSION: The DC% of SAE implants contaminated with E coli or P gingivalis by means of chemicals commonly used in dentistry is high, with the exception of Ca(OH)2 paste burnished for 1 minute and then irrigated with saline solution for 1 minute.