Cryo-EM Structure of Porphyromonas gingivalis RNA Polymerase.

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in ß and ß' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development.

Host and bacterial factors linking periodontitis and rheumatoid arthritis.

Observations from numerous clinical, epidemiological and serological studies link periodontitis with severity and progression of rheumatoid arthritis. The strong association is observed despite totally different aetiology of these two diseases, periodontitis being driven by dysbiotic microbial flora on the tooth surface below the gum line, while rheumatoid arthritis being the autoimmune disease powered by anti-citrullinated protein antibodies (ACPAs). Here we discuss genetic and environmental risk factors underlying development of both diseases with special emphasis on bacteria implicated in pathogenicity of periodontitis. Individual periodontal pathogens and their virulence factors are argued as potentially contributing to putative causative link between periodontal infection and initiation of a chain of events leading to breakdown of immunotolerance and development of ACPAs. In this respect peptidylarginine deiminase, an enzyme unique among prokaryotes for Porphyromonas gingivalis, is elaborated as a potential mechanistic link between this major periodontal pathogen and initiation of rheumatoid arthritis development.

Expanded substrate specificity supported by P1' and P2' residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides.

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

Porphyromonas gingivalis exacerbates ulcerative colitis via Porphyromonas gingivalis peptidylarginine deiminase.

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.

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.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Structural and kinetic characterization of Porphyromonas gingivalis glutaminyl cyclase.

Porphyromonas gingivalis is a bacterial species known to be involved in the pathogenesis of chronic periodontitis, that more recently has been as well associated with Alzheimer's disease. P. gingivalis expresses a glutaminyl cyclase (PgQC) whose human ortholog is known to participate in the beta amyloid peptide metabolism. We have elucidated the crystal structure of PgQC at 1.95 Å resolution in unbound and in inhibitor-complexed forms. The structural characterization of PgQC confirmed that PgQC displays a mammalian fold rather than a bacterial fold. Our biochemical characterization indicates that PgQC uses a mammalian-like catalytic mechanism enabled by the residues Asp149, Glu182, Asp183, Asp218, Asp267 and His299. In addition, we could observe that a non-conserved Trp193 may drive differences in the binding affinity of ligands which might be useful for drug development. With a screening of a small molecule library, we have identified a benzimidazole derivative rendering PgQC inhibition in the low micromolar range that might be amenable for further medicinal chemistry development.

PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two ß-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the "attachment complex" composed of PorU, PorZ, and the integral OM ß-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

PPAD Activity Promotes Outer Membrane Vesicle Biogenesis and Surface Translocation by Porphyromonas gingivalis.

Many bacteria switch between a sessile and a motile mode in response to environmental and host-related signals. Porphyromonas gingivalis, an oral anaerobe implicated in the etiology of chronic periodontal disease, has long been described as a nonmotile bacterium. And yet, recent studies have shown that under certain conditions, P. gingivalis is capable of surface translocation. Considering these findings, this work aimed to increase our understanding of how P. gingivalis transitions between sessile growth and surface migration. Here, we show that the peptidylarginine deiminase secreted by P. gingivalis (PPAD), an enzyme previously shown to be upregulated during surface translocation and to constrain biofilm formation, promotes surface translocation. In the absence of PPAD, the production of outer membrane vesicles (OMVs) was drastically reduced. In turn, there was a reduction in gingipain-mediated proteolysis and a reduced zone of hydration around the site of inoculation. Transcriptome sequencing (RNA-Seq) and metabolomics analyses also showed that these changes corresponded to a shift in arginine metabolism. Overall, this report provides new evidence for the functional relevance of PPAD and proteases, as well as the importance of PPAD activity in OMV biogenesis and release. Our findings support the model that citrullination is a critical mechanism during lifestyle transition between surface-attached growth and surface translocation by modulating OMV-mediated proteolysis and arginine metabolism.IMPORTANCE Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production, and many of the proteins packaged in these vesicles are proteolytic or protein-modifying enzymes. This includes key virulence determinants, such as the gingipains and PPAD (a unique peptidylarginine deiminase). Here, we show that PPAD activity (citrullination) is involved in OMV biogenesis. The study revealed an unusual mechanism that allows this bacterium to transform its surroundings. Since OMVs are detected in circulation and in systemic tissues, our study results also support the notion that PPAD activity may be a key factor in the correlation between periodontitis and systemic diseases, further supporting the idea of PPAD as an important therapeutic target.

Manipulation of Saliva-Derived Microcosm Biofilms To Resemble Dysbiotic Subgingival Microbiota.

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and reestablish a health-associated microbiota, a high-throughput in vitro multispecies biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rRNA gene amplicon sequencing as well as measurement of dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principal-component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis to saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder a microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies.IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed targeting reversing dysbiosis and restoring host-compatible microbiota rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in a dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large-scale evaluation of strategies that can potentially modulate microbial ecology.

Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics.

Background:Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation. Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots. Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting. Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains.

Uric acid is a potential metabolite that serves as a danger-associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP-1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase-1 activation, cell death, and the expression of proinflammatory cytokines, including IL-1α, IL-6, and IL-8, in the human keratinocyte HOK-16B cell line. Our results suggest that gingipain-induced uric acid can mediate inflammation in periodontal tissue cells.

Targeting a cysteine protease from a pathobiont alleviates experimental arthritis.

BACKGROUND: Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of auto-inflammatory diseases. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant bone loss in model systems and in patients. OBJECTIVE: We sought to gain further insight into the role of this pathobiont in rheumatoid arthritis (RA) and to identify novel therapeutic targets for auto-inflammatory diseases. METHODS: We profiled the antibody response to RgPA-specific domains in patient sera. We also tested the potential protective effects of RgpA domains in an experimental arthritis model. RESULTS: Pre-immunization of rats with purified recombinant RgpA domains alleviated arthritis in the joints of the rodents and reduced bone erosion. Using a functional genomics approach at both the mRNA and protein levels, we report that the pre-immunizations reduced arthritis severity by impacting a matrix metalloprotease characteristic of articular injury, a chemokine known to be involved in recruiting inflammatory cells, and three inflammatory cytokines. Finally, we identified an amino acid motif in the RgpA catalytic domain of P. gingivalis that shares sequence homology with type II collagen. CONCLUSION: We conclude that pre-immunization against gingipain domains can reduce the severity of experimentally induced arthritis. We suggest that targeting gingipain domains by pre-immunization, or, possibly, by small-molecule inhibitors, could reduce the potential of P. gingivalis to translocate to remote tissues and instigate and/or exacerbate pathology in RA, but also in other chronic inflammatory diseases.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen.

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.

Gingimaps: Protein Localization in the Oral Pathogen Porphyromonas gingivalis.

Porphyromonas gingivalis is an oral pathogen involved in the widespread disease periodontitis. In recent years, however, this bacterium has been implicated in the etiology of another common disorder, the autoimmune disease rheumatoid arthritis. Periodontitis and rheumatoid arthritis were known to correlate for decades, but only recently a possible molecular connection underlying this association has been unveiled. P. gingivalis possesses an enzyme that citrullinates certain host proteins and, potentially, elicits autoimmune antibodies against such citrullinated proteins. These autoantibodies are highly specific for rheumatoid arthritis and have been purported both as a symptom and a potential cause of the disease. The citrullinating enzyme and other major virulence factors of P. gingivalis, including some that were implicated in the etiology of rheumatoid arthritis, are targeted to the host tissue as secreted or outer-membrane-bound proteins. These targeting events play pivotal roles in the interactions between the pathogen and its human host. Accordingly, the overall protein sorting and secretion events in P. gingivalis are of prime relevance for understanding its full disease-causing potential and for developing preventive and therapeutic approaches. The aim of this review is therefore to offer a comprehensive overview of the subcellular and extracellular localization of all proteins in three reference strains and four clinical isolates of P. gingivalis, as well as the mechanisms employed to reach these destinations.

Novel synthetic peptide derived from Porphyromonas gingivalis Lys-gingipain detects IgG-mediated host response in periodontitis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis.

A Potential Role of Phospholipase 2 Group IIA (PLA2-IIA) in P. gingivalis-Induced Oral Dysbiosis.

Porphyromonas gingivalis is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which P. gingivalis could abrogate the host-microbe symbiotic relationship leading to oral dysbiosis remain unclear. We have recently demonstrated that P. gingivalis specifically increased the antimicrobial properties of oral epithelial cells, through a strong induction of the expression of PLA2-IIA in a mechanism that involves activation of the Notch-1 receptor. Moreover, gingival expression of PLA2-IIA was significantly increased during initiation and progression of periodontal disease in non-human primates and interestingly, those PLA2-IIA expression changes were concurrent with oral dysbiosis. In this chapter, we present an innovative hypothesis of a potential mechanism involved in P. gingivalis-induced oral dysbiosis and inflammation based on our previous observations and a robust body of literature that supports the antimicrobial and proinflammatory properties of PLA2-IIA as well as its role in other chronic inflammatory diseases.

Potential Oral Health Care Agent from Coffee Against Virulence Factor of Periodontitis.

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.

Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase.

Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.

Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host-pathogen interactions.

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.