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Background An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. Methods We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis ( P. gingivalis ) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. Results Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. Conclusions Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

There is accumulating data suggesting that periodontitis is associated with increased risk of systemic and autoimmune diseases, including cardiovascular disease, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and there is an unmet need to identify these individuals early. With the periodontal bacteria Porphyromonas gingivalis (Pg) as one of the key drivers of periodontitis, we set out to investigate whether antibodies to Pg virulence factor arginine gingipain (Rgp) could serve as a biomarker for periodontitis patients at increased risk of autoimmunity and systemic disease. We measured serum anti-Rgp IgG in three study populations: PAROKRANK (779 individuals with myocardial infarction (MI); 719 controls), where 557 had periodontitis, and 312 were positive for autoantibodies associated with RA/SLE; the PerioGene North pilot (41 periodontitis; 39 controls); and an SLE case/control study (101 SLE; 100 controls). Anti-Rgp IgG levels were increased in severe periodontitis compared to controls (p < 0.0001), in individuals positive for anti-citrullinated protein antibodies (p = 0.04) and anti-dsDNA antibodies (p = 0.035), compared to autoantibody-negative individuals; and in MI patients versus matched controls (p = 0.035). Our data support longitudinal studies addressing the role of anti-Rgp antibodies as biomarkers for periodontitis patients at increased risk of developing autoimmunity linked to RA and SLE, and mechanisms underpinning these associations.

There is accumulating data suggesting that periodontitis is associated with increased risk of systemic and autoimmune diseases, including cardiovascular disease, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and there is an unmet need to identify these individuals early. With the periodontal bacteria Porphyromonas gingivalis (Pg) as one of the key drivers of periodontitis, we set out to investigate whether antibodies to Pg virulence factor arginine gingipain (Rgp) could serve as a biomarker for periodontitis patients at increased risk of autoimmunity and systemic disease. We measured serum anti-Rgp IgG in three study populations: PAROKRANK (779 individuals with myocardial infarction (MI); 719 controls), where 557 had periodontitis, and 312 were positive for autoantibodies associated with RA/SLE; the PerioGene North pilot (41 periodontitis; 39 controls); and an SLE case/control study (101 SLE; 100 controls). Anti-Rgp IgG levels were increased in severe periodontitis compared to controls (p < 0.0001), in individuals positive for anti-citrullinated protein antibodies (p = 0.04) and anti-dsDNA antibodies (p = 0.035), compared to autoantibody-negative individuals; and in MI patients versus matched controls (p = 0.035). Our data support longitudinal studies addressing the role of anti-Rgp antibodies as biomarkers for periodontitis patients at increased risk of developing autoimmunity linked to RA and SLE, and mechanisms underpinning these associations.

Porphyromonas gingivalis is a major pathogenic bacterium involved in the pathogenesis of periodontitis. Citrullination has been reported as the underlying mechanism of the pathogenesis, which relies on the interplay between two virulence factors of the bacterium, namely gingipain R and the bacterial peptidyl arginine deiminase. Gingipain R cleaves host proteins to expose the C-terminal arginines for peptidyl arginine deiminase to citrullinate and generate citrullinated proteins. Apart from carrying out citrullination in the periodontium, the bacterium is found capable of citrullinating proteins present in the host synovial tissues, atherosclerotic plaques and neurons. Studies have suggested that both virulence factors are the key factors that trigger distal effects mediated by citrullination, leading to the development of some non-communicable diseases, such as rheumatoid arthritis, atherosclerosis, and Alzheimer’s disease. Thus, inhibition of these virulence factors not only can mitigate periodontitis, but also can provide new therapeutic solutions for systematic diseases involving bacterial citrullination. Herein, we described both these proteins in terms of their unique structural conformations and biological relevance to different human diseases. Moreover, investigations of inhibitory actions on the enzymes are also enumerated. New approaches for identifying inhibitors for peptidyl arginine deiminase through drug repurposing and virtual screening are also discussed.

Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model. The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice. The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Epidemiologic studies have demonstrated a link between periodontitis (PD) and rheumatoid arthritis (RA), specifically RA characterized by anti-citrullinated protein antibodies (ACPA). The keystone pathogen driving PD, Porphyromonas gingivalis (Pg), is the only pathogen known to express peptidylarginine deiminase (PAD), a citrullinating enzyme. Hence, Pg has been proposed to be involved in triggering the ACPA response, by generating citrullinated antigens in an inflammatory milieu(1). Another major virulence factor of Pg is arginine gingipain B (RgpB), a proteinase which cleaves proteins so that P.PAD can access the site where citrullination takes place. We have previously shown elevated anti-RgpB IgG levels in ACPA+ RA patients, even before clinical onset(2, 3), and we hypothesize that anti-RgpB IgG could serve as a serological marker to identify PD patients with increased risk of developing ACPA+ RA. Based on this hypothesis, we set out to investigate whether anti-RgpB IgG was associated with PD, PD severity, autoimmunity in general, and the ACPA response in particular. Anti-RgpB IgG, as well as RA- and systemic lupus erythematosus (SLE)-related autoantibodies targeting cyclic citrullinated peptide(s) (CCP2), rheumatoid factor (RF), dsDNA, cardiolipin, and β2 glycoprotein, were measured by ELISA in serum samples from the ParoKrank study, which is a well-characterized cohort of 805 patients with a first myocardial infarction and 805 matched controls, where periodontal status has been determined by dentists(4). In this study, individuals with PD (n=941) were compared to individuals without PD (n=557). We detected significantly elevated (p<0,0001) anti-RgpB IgG levels in PD compared to non-PD individuals, with highest levels recorded in severe PD. Anti-RgpB IgG levels were significantly increased in PD patients positive for CCP2 and/or RF (n=50), when compared to PD patients negative for CCP2 and RF (n=507), p<0,05, and when compared to non-PD individuals positive for CCP2 and/or RF (n=62), p < 0,05. Notably, these differences were not seen for SLE-related autoantibodies. In addition, anti-RgpB IgG levels were significantly elevated amongst MI patients versus controls (p < 0,05), supporting the previous finding that PD is more common among MI patients(4). Our data demonstrates a specific association between severe PD, elevated anti-RgpB IgG levels and RA-related autoantibodies, supporting a role for Pg in linking PD to ACPA+ RA. Further investigation will be needed to confirm whether anti-RgpB IgG can be used as a serological marker to identify PD patients with increased risk of developing ACPA+ RA. [1]Rosenstein ED, Greenwald RA, Kushner LJ, Weissmann G. Hypothesis: the humoral immune response to oral bacteria provides a stimulus for the development of rheumatoid arthritis. Inflammation. 2004;28(6):311-8. [2]Kharlamova N, Jiang X, Sherina N, Potempa B, Israelsson L, Quirke AM, et al. Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology. Arthritis Rheumatol. 2016;68(3):604-13. [3]Johansson L, Sherina N, Kharlamova N, Potempa B, Larsson B, Israelsson L, et al. Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis. Arthritis Res Ther. 2016;18(1):201. [4]Rydén L, Buhlin K, Ekstrand E, Faire Ud, Gustafsson A, Holmer J, et al. Periodontitis Increases the Risk of a First Myocardial Infarction. Circulation. 2016;133(6):576-83. None declared

Periodontitis is a global health problem and the 6 th most common infectious disease worldwide. Porphyromonas gingivalis is considered a keystone pathogen in the disease and is capable of elevating the virulence potential of the periodontal microbial community. Strategies that interfere with P. gingivalis colonization and expression of virulence factor are therefore attractive approaches for preventing and treating periodontitis. We have previously reported that an 11-mer peptide (SAPP) derived from Streptococcus cristatus arginine deiminase (ArcA) was able to repress the expression and production of several well-known P . gingivalis virulence factors including fimbrial proteins and gingipains. Herein we expand and develop these studies to ascertain the impact of this peptide on phenotypic properties of P. gingivalis related to virulence potential. We found that growth rate was not altered by exposure of P. gingivalis to SAPP, while monospecies and heterotypic biofilm formation, and invasion of oral epithelial cells were inhibited. Additionally, SAPP was able to impinge the ability of P. gingivalis to dysregulate innate immunity by repressing gingipain-associated degradation of interleukin-8 (IL8). Hence, SAPP has characteristics that could be exploited for the manipulation of P. gingivalis levels in oral communities and preventing realization of virulence potential.

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.

P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.

ObjectivesThe 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 methodsRecombinant 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.ResultsMALDI-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.ConclusionsUsing 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 relevanceThe 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.