Differences in virulence within the species Bacteroides gingivalis

In order to gain insight into the relative importance of several virulence factors of Bacteroides gingivalis, 8 strains with a varying virulence were studied. The virulence of B. gingivalis was determined in a mouse model. Strains HG 66, HG 76 and HG 184 were very virulent causing phlegmonos abscesses with lesions and necrosis. The strains HG 405 and HG 462 caused phlegmonous abscesses with pus. Strains HG 91, HG 94 and HG 185 were less virulent and induced gravity abscesses. In vitro strains HG 66, HG 76 and HG 184 induced low amounts of chemiluminescence by polymorphonuclear leucocytes. All other strains including HG 405 and HG 462 caused a relatively high chemiluminescence. Most strains displayed a high sensitivity to the bactericidal activity of fresh serum except for the highly virulent strains HG 66, HG 76 and HG 184. No differences in extracellular proteolytic activity on Azocoll, production of volatile fatty acids and ammonia were found between the B. gingivalis strains studied. In conclusion, differences in virulence were shown within the species B. gingivalis; the relative importance of several virulence factors was investigated.     

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis,a target for improved oral health

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.     

Bacteroides gingivalis,Bacteroides asaccharolyticus,andBacteroides melaninogenicus subspecies: Cell surface morphology and adherence to erythrocytes and human buccal epithelial cells

All study strains ofBacteroides gingivalis, B. asaccharolyticus, andB. melaninogenicus subspecies possessed numerous pilus-like fibers and capsule-like outer surface structures. The capsular morphology varied between the different species and subspecies.B. gingivalis strongly agglutinated 16 erythrocyte species studied.B. asaccharolyticus showed variable and weak agglutination of only a few erythrocyte species.B. melaninogenicus subsp.intermedius strains strongly agglutinated rabbit erythrocytes and exhibited variable, often weak agglutination of 8 other erythrocyte species. Preparations of capsular polysaccharide or lipopolysaccharide fromB. gingivalis failed to agglutinate human erythrocytes, while pili preparations from the same organisms possessed marked hemagglutinating activity.B. gingivalis cells adhered in high numbers to human buccal epithelial cells, whereas strains ofB. asaccharolyticus failed to show measurable adherence. Oral strains ofB. melaninogenicus subsp.intermedius feebly adhered to the buccal epithelial cells. Pretreatment ofB. gingivalis cells with serum or saliva prevented the adherence to epithelial cells. Our findings suggest that cell surfaces with distinct properties exist on the various black-pigmentedBacteroides species and subspecies and this may accout for markedly differing ability of these organisms to attach to mammalian cells.     

Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells

The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.     

Research on the roles of genes coding ATP-binding cassette transporters in Porphyromonas gingivalis pathogenicity

Porphyromonas gingivalis, as a major pathogen of periodontitis, could rapidly adhere to and invade host gingival epithelial cells (GECs) for the induction of infection. One ATP-binding cassette (ABC) transporter gene was found to be upregulated during this infection process, however, the molecular mechanisms remain unclear. In this study, we systemically investigated the messenger RNA level changes of all ABC transporter family genes in P. gingivalis while being internalized within GECs by real-time polymerase chain reaction. We identified that two ABC transporter genes, PG_RS04465 (PG1010) and PG_RS07320 (PG1665), were significantly increased in P. gingivalis after coculturing with GECs. Mutant strains with knockout (KO) of these two genes were generated by homogenous recombination. PG_RS04465 and PG_RS07320 KO mutants showed no change in the growth of bacteria per se. Knockdown of PG_RS07320, but not PG_RS04465, caused decreased endotoxin level in the bacteria. In contrast, both mutant strains showed decreased Arg- and Lys-gingipains activities, with significantly reduced adhesion and invasion capabilities. Secreted interleukin-1β (IL-1β) and IL-6 levels in GECs cocultured with PG_RS04465 or PG_RS07320 KO mutants were also decreased, whereas, only the cells cocultured with PG_RS07320 KO mutants showed significant decrease. In addition, virulence study using mouse revealed that both KO mutant strains infection caused less mouse death than wild-type strains, showing reduced virulence of two KO strains. These results indicated that ABC transporter genes PG_RS04465 and PG_RS07320 are positive regulators of the virulence of P. gingivalis.     

Peptide Mapping of a Functionally Versatile Fimbrial Adhesin from <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Porphyromonas gingivalis is strongly implicated in adult periodontitis. This oral pathogen expresses adhesive filamentous appendages, known as fimbriae, which constitute one of its major virulence factors. Fimbriae are composed of polymerized fimbrillin (FimA) subunits and play an indispensable role in the ability of P. gingivalis to colonize and invade periodontal tissue and to induce alveolar bone loss. The virulence potential of fimbriae is attributable to their capacity to interact with various dental or epithelial substrates, extracellular matrix proteins, other bacteria, and host immune cells. It has been puzzling whether the multifunctional adhesive ability of fimbriae results from multiple adhesion epitopes specific for each receptor, or whether fimbriae contain versatile structural motifs that are recognizable by multiple receptors. This review summarizes peptide mapping studies that have defined functional epitopes of P. gingivalis fimbriae. Available evidence suggests that the binding of fimbriae to various receptors generally involves specific amino acid sequences of the FimA subunit, although the same FimA peptide may occasionally recognize different receptors. Moreover, in cases where distinct FimA peptides interact with the same receptor, the peptides involved share common sequences. It therefore appears that the promiscuous binding reactivity of P. gingivalis fimbriae is attributable to a multitude of adhesion epitopes which however share minimal binding elements, although the overall hydrophobicity and polymeric nature of fimbriae may significantly enhance the avidity of binding interactions. Peptide mapping of fimbriae is significant also for translational purposes, such as for development of subunit vaccines that contain defined immunogenic and functionally important epitopes and for identification of peptides that can competitively inhibit virulence activities of P. gingivalis fimbriae. Studies performed in the author’s lab and cited in this review were supported by U.S. Public Health Service Grant DE015254 from the NIDCR, National Institutes of Health.     

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.     

Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis

Background  

Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.     

Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases

Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 μg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis.     

Periodontitis,periodontopathic bacteria and lactoferrin

Lactoferrin (LF) is a component of saliva and is suspected to be a defense factor against oral pathogens including Streptococcus mutans and Candida albicans. Periodontitis is a very common oral disease caused by periodontopathic bacteria. Antimicrobial activities and other biological effects of LF against representative periodontopathic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, have been widely studied. Association of polymorphisms in LF with incidence of aggressive periodontitis and the role of LF in the gingival crevicular fluid as a marker of periodontitis severity have also been reported. Periodontopathic bacteria reside as a biofilm in supragingival and subgingival plaque. Our recent study indicated that LF exhibits antibacterial activity against planktonic forms of P. gingivalis and P. intermedia at higher concentrations, and furthermore, LF effectively inhibits biofilm formation and reduces the established biofilm of these bacteria at physiological concentrations. A small-scale clinical study indicated that oral administration of bovine LF reduces P. gingivalis and P. intermedia in the subgingival plaque of chronic periodontitis patients. LF seems to be a biofilm inhibitor of periodontopathic bacteria in vitro and in vivo.     

An experimentally induced phlegmonous abscess by a strain of Bacteroides gingivalis in guinea pigs and mice

The virulence of B. gingivalis strain W83 was studied in an experimental animal model. Cells grown overnight, washed and resuspended in broth, were injected intradermally or subcutaneously in the back of guinea pigs, rats and mice. This strain proved to be very virulent, causing a severe plegmonous abscess in guinea pigs. Also in mice, which are thought to be resistant to infections with black-pigmented Bacteroides strains, the same type of infection could be induced. Rats proved to be rather insensitive. The model presented can be used as a simple virulence test for these anaerobic bacteria.     

The capsule of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> reduces the immune response of human gingival fibroblasts

Background  

Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS). Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

Porphyromonas gingivalis induces the production of interleukin‐31 by human mast cells,resulting in dysfunction of the gingival epithelial barrier

Interleukin (IL)‐31 is important for innate immunity in mucosal tissues and skin, and increased IL‐31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL‐31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL‐31 expression in the gingival tissues of wild‐type mice but not in those of mast cell‐deficient mice. The P. gingivalis‐induced IL‐31 production by human mast cells occurred through the activation of the JNK and NF‐κB signalling pathways and was dependent on the P. gingivalis lysine‐specific protease gingipain‐K. P. gingivalis infection induced IL‐31 receptor α and oncostatin M receptor β expression in human gingival epithelial cells. Notably, the P. gingivalis‐induced IL‐31 production by mast cells led to the downregulation of claudin‐1, a tight junction molecule, in gingival epithelial cells, resulting in an IL‐31‐dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL‐31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.     

Porphyromonas gingivalis GroEL Induces Osteoclastogenesis of Periodontal Ligament Cells and Enhances Alveolar Bone Resorption in Rats

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.     

Pyocycanin,a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.     

Regulation of ICAM‐1 expression in gingival fibroblasts infected with high‐glucose‐treated P. gingivalis

Porphyromonas gingivalis is a major pathogen in the initiation and progression of periodontal disease, which is recognized as a common complication of diabetes. ICAM‐1 expression by human gingival fibroblasts (HGFs) is crucial for regulating local inflammatory responses in inflamed periodontal tissues. However, the effect of P. gingivalis in a high‐glucose situation in regulating HGF function is not understood. The P. gingivalis strain CCUG25226 was used to study the mechanisms underlying the modulation of HGF ICAM‐1 expression by invasion of high‐glucose‐treated P. gingivalis (HGPg). A high‐glucose condition upregulated fimA mRNA expression in P. gingivalis and increased its invasion ability in HGFs. HGF invasion with HGPg induced increases in the expression of ICAM‐1. By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of p38 MAPK and Akt pathways is critical for HGPg‐induced ICAM‐1 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that HGPg invasion increases NF‐κB‐ and Sp1‐DNA‐binding activities in HGFs. Inhibition of NF‐κB and Sp1 activations blocked the HGPg‐induced ICAM‐1 promoter activity and expression. The effect of HGPg on HGF signalling and ICAM‐1 expression is mediated by CXC chemokine receptor 4 (CXCR4). Our findings identify the molecular pathways underlying HGPg‐dependent ICAM‐1 expression in HGFs, providing insight into the effect of P. gingivalis invasion in HGFs.     

The core genome of the anaerobic oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Background  

The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.     

Species specificity,surface exposure,protein expression,immunogenicity, and participation in biofilm formation of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> HmuY

Background  

Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY.