Search

Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 μg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 μM), samarangenin B (71%, 20 μM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 μM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC 50 12.78 μg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC 50 3 μg/mL). On Lysine-specific gingipain, AQF has an IC 50 15.89, EAF 14.15, and FLB7 6 μg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC 50 11.34 (AQF), 11.66 (EAF), and 12.09 μg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg., Competing Interests: Declaration of competing interest This study was financially supported by CNPq, for providing a student grant to Raquel Isolani for the program “Science Without Borders” – CNPq (Grant #200173/2018–9). Residual expenses were borne by intramural annual budgets from the University of Münster to Andreas Hensel, the State University of Maringá to and project's funding agencies, CAPES, FINEP, CNPq to João Carlos Palazzo de Mello (Grant #401490/2014-9). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis., Materials and Methods: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides., Results: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin., Conclusions: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides., Clinical Relevance: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.

Background and Objective: Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease., Material and Methods: Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of gingipains were examined using purified gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids., Results: We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited gingipain activity in a non-competitive manner with K i values at μm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of gingipain inhibition by the compounds., Conclusion: The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses gingipains, P. gingivalis growth and biofilm formation., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD., (© FASEB.)

Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection., (2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd)

Background: Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments., Methods: Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis., Results: PDRgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2nM to 0.85nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PDRgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex., Conclusion: Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains., General Significance: Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bacterial biofilms can be viewed as a specific type of persistent bacterial infection. After initial invasion, microbes can attach to living and non-living surfaces, such as prosthetics and indwelling medical devices, and form a biofilm composed of extracellular polysaccharides, proteins, and other components. In hosts, biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. The clinical aspects of biofilm formation and leading strategies for biofilm inhibitors will be discussed in this mini-review.

Porphyromonas gingivalis strains W50 and ATCC 33277 were shown to bind to cultured human fibroblast (MRC-5) cells using flow cytometry. As the concentration of P. gingivalis strain W50 cells was increased relative to the concentration of MRC-5 cells, the number of W50 cells bound per MRC-5 cell increased, as did the percentage of MRC-5 cells with bacteria bound. However, this relationship was only seen for P. gingivalis strain ATCC 33277 at low cell concentrations: at high bacterial cell concentrations strain ATCC 33277 auto-aggregated and binding to the MRC-5 cells decreased. Strain W50 was therefore chosen to study the role of the surface proteinase-adhesin complexes (RgpA-Kgp complexes) in binding to MRC-5 cells. P. gingivalis W50 cells treated with an inhibitor of the RgpA-Kgp complexes exhibited reduced binding to MRC-5 cells. The purified active and proteinase-inactive RgpA-Kgp complexes competitively inhibited binding of W50 to MRC-5 cells, and isogenic mutants of W50 lacking RgpA/B and Kgp displayed reduced binding. P. gingivalis W50 mutant cells lacking Kgp exhibited the lowest binding to MRC-5 cells, suggesting an important role for this proteinase and its associated adhesins in binding to fibroblasts.

Porphyromonas gingivalis gingipains is suspected to be one of the most important causative agents of periodontitis. We postulated that the inhibition of gingipains may reduce the pathogenic nature of P. gingivalis. Anti-P. gingivalis egg yolk antibody (IgY-GP) was isolated from the yolks of hens immunized with purified gingipains. We applied IgY-GP gel subgingivally in periodontitis patients who harbored P. gingivalis in their subgingival flora. Five pairs of contralateral anterior single-rooted teeth were selected. One tooth in each contralateral pair was randomly treated with IgY-GP and subgingival scaling and root planing, whereas the other tooth was treated with SRP alone. The number of P. gingivalis bacteria was assessed by real-time PCR. Bacterial levels were expressed as the percentage of total bacteria. The IgY-GP group had a significant reduction in probing depth. BOP significantly decreased in the IgY-GP group compared to the control group at week 4. The levels of P. gingivalis significantly increased in the control group at week 4, whereas the reduction in the levels of P. gingivalis was sustained in the IgY-GP group. Within the limitations of the present study, IgY-GP was shown to be an effective immunotherapeutic agent in the treatment of periodontitis.

We have shown previously that gingipains from Porphyromonas gingivalis W83 can induce cell detachment, cell adhesion molecule (CAM) cleavage, and apoptosis in endothelial cells; however, the specific roles of the individual gingipains are unclear. Using purified gingipains, we determined that each of the gingipains can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells. Interestingly, in the absence of active caspases, both gingipain-active W83 extracts and purified HRgpA and RgpB induce apoptotic morphology, suggesting that the gingipains can induce both caspase-dependent and caspase-independent apoptosis. Using z-VAD-FMK to inhibit Kgp activity and leupeptin to inhibit Rgp activity in gingipain-active W83 extracts, we investigated the relative significance of the synergistic role of the gingipains. z-VAD-FMK or leupeptin delayed, but did not inhibit, cell detachment induced by gingipain-active W83 extracts or purified gingipains. There was partial cleavage of N-cadherin and cleavage of VE-cadherin was not inhibited. Degradation of integrin beta1 was inhibited only in the presence of z-VAD-FMK. These results further clarify the role P. gingivalis plays in tissue destruction occurring in the periodontal pocket.

Background/aims: Gingipains, proteolytic enzymes produced by the periodontal pathogen Porphyromonas gingivalis, are regarded as virulence factors in the pathogenesis of periodontitis. Inhibition of gingipain activity therefore may have therapeutic potential, and it has been suggested that chlorhexidine may inhibit the activities of these enzymes. The purposes of the present study were to examine systematically the inhibitory effects of chlorhexidine on three purified gingipains and to determine the effect of Zn(II) on chlorhexidine inhibition., Methods: The activities of lys-gingipain (Kgp) and two forms of arg-gingipain (RgpB and HRgpA) were measured in the presence of varying concentrations of chlorhexidine and with chlorhexidine supplemented with Zn(II). Inhibition constants (K(i)'s) were determined for chlorhexidine alone and in the presence of Zn(II). Fractional inhibitory constant indices were calculated to assess the synergy of the chlorhexidine-Zn(II) inhibition., Results: RgpB, HRgpA, and Kgp were all inhibited by chlorhexidine with K(i)'s in the micromolar range. For RgpB and HRgpA, the inhibitory effects of chlorhexidine were enhanced 3-30-fold by Zn(II). The chlorhexidine-Zn(II) interaction was synergistic for inhibition of HRgpA and RgpB. For Kgp, the effect of Zn(II) on chlorhexidine inhibition was antagonistic., Conclusions: Chlorhexidine is an effective inhibitor of gingipains, and the inhibition of R-gingipains is enhanced by Zn(II). A mixture of chlorhexidine and Zn(II) may be useful as an adjunct in the treatment of periodontitis and in the post-treatment maintenance of periodontitis patients.

Objective: Arginine-specific cysteine proteases (Rgps) from Porphyromonas gingivalis are important virulent factors of periodontal diseases. However, there is no therapeutic drug that inhibits proinflammatory events induced by these enzymes. In this study, we investigated proinflammatory activities of Rgps and activated coagulation factor X (FXa) and examined the effect of DX-9065a, a new selective inhibitor of FXa, on proinflammatory events induced by these proteinases., Methods: Human gingival fibroblasts were stimulated with Rgps and FXa in the presence or absence of DX-9065a, and then interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1) release, their mRNA expression, and nuclear factor kappaB (NF-kappaB) activation were assessed using an enzyme-linked immunosorbent assay (ELISA), northern blotting, and a gel-mobility shift method, respectively., Results: Rgps and FXa activated IL-6 and MMP-1 release in human gingival fibroblasts through their amidolytic activities and in mitogen-activated protein kinase (MAPK) and NF-kappaB dependent manners. DX-9065a inhibited FXa-induced IL-6 mRNA expression and NF-kappaB activation. DX-9065a inhibited amidolytic activities of FXa and Rgps in vitro and ex vivo., Conclusion: Rgps and FXa are potent inflammatory mediators and DX-9065a may be a useful therapeutic drug for periodontal disease.

In this study, we developed a procedure to produce gingivitis in rats by inoculation of Porphyromonas gingivalis and studied the contribution of the bacterial cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp), to the pathology in the gingiva. To adhere the bacterium to periodontal tissues, a cotton thread was inserted between the first and second molar of right maxillary sites of rats. Rats in group A were administered with vehicle alone after bacterial (strain W83) inoculation. In group B, the bacteria were inoculated in combination with leupeptin, a potent inhibitor of Rgp and Kgp, and then leupeptin alone was administered the week after. Rats in group C were administered leupeptin for 6 weeks after bacteria inoculation. All left maxillary gingiva in three groups showed no inflammatory changes. Right maxillary gingiva of group A showed most of the clinical landmarks of gingivitis. Leupeptin exhibited only a little inhibitory effect on this gingivitis in group B, whereas it had a strong inhibitory effect on the inflammation in group C. These results suggest that P. gingivalis-induced gingivitis is attributable to Rgp and Kgp and that leupeptin is more effective in the late phase than the early stage of gingivitis.