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Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer's. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-κB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection. [ABSTRACT FROM AUTHOR]

Cell death is a natural consequence of infection. However, although the induction of cell death was solely thought to benefit the pathogen, compelling data now show that the activation of cell death pathways serves as a nuanced antimicrobial strategy that couples pathogen elimination with the generation of inflammatory cytokines and the priming of innate and adaptive cellular immunity. Following cell death, the phagocytic uptake of the infected dead cell by antigen‐presenting cells and the subsequent lysosomal fusion of the apoptotic body containing the pathogen serve as an important antimicrobial mechanism that furthers the development of downstream adaptive immune responses. Despite the complexity of regulated cell death pathways, pathogens are highly adept at evading them. Here, we provide an overview of the remarkable diversity of cell death and efferocytic pathways and discuss illustrative examples of virulence strategies employed by pathogens, including oral pathogens, to counter their activation and persist within the host. [ABSTRACT FROM AUTHOR]

Patients with rheumatoid arthritis (RA) have altered levels of exhaled nitric oxide (NO) compared with healthy controls. Here, we investigated whether the clinical features of and immunological factors in RA pathogenesis could be linked to the NO lung dynamics in early disease. A total of 44 patients with early RA and anti-citrullinated peptide antibodies (ACPAs), specified as cyclic citrullinated peptide 2 (CCP2), were included. Their exhaled NO levels were measured, and the alveolar concentration, the airway compartment diffusing capacity and the airway wall concentration of NO were estimated using the Högman–Meriläinen algorithm. The disease activity was measured using the Disease Activity Score for 28 joints. Serum samples were analysed for anti-CCP2, rheumatoid factor, free secretory component, secretory component containing ACPAs, antibodies against Porphyromonas gingivalis (Rgp) and total levels of IgA, IgA1 and IgA2. Significant negative correlations were found between the airway wall concentration of NO and the number of swollen joints (Rho −0.48, p = 0.004), between the airway wall concentration of NO and IgA rheumatoid factor (Rho −0.41, p = 0.017), between the alveolar concentration and free secretory component (Rho −0.35, p = 0.023) and between the alveolar concentration and C-reactive protein (Rho −0.36, p = 0.016), but none were found for anti-CCP2, IgM rheumatoid factor or the anti-Rgp levels. In conclusion, altered NO levels, particularly its production in the airway walls, may have a role in the pathogenesis of ACPA-positive RA. [ABSTRACT FROM AUTHOR]

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis. [ABSTRACT FROM AUTHOR]

Periodontal disease is one of the most common forms of inflammation. It is currently diagnosed by observing symptoms such as gingival bleeding and attachment loss. However, the detection of biomarkers that precede such symptoms would allow earlier diagnosis and prevention. Aptamers are short oligonucleotides or peptides that fold into three‐dimensional conformations conferring the ability to bind molecular targets with high affinity and specificity. Here we report the selection of aptamers that bind specifically to the bacterium Tannerella forsythia, a pathogen frequently associated with periodontal disease. Two aptamers with the highest affinity were examined in more detail, revealing that their binding is probably dependent on mirolysin, a surface‐associated protease secreted by the T. forsythia type‐9 secretion system. The aptamers showed minimal cross‐reactivity to other periodontopathogens and are therefore promising leads for the development of new tools to study the composition of the periodontitis‐associated dysbiotic bacteriome as well as inexpensive new diagnostic assays. [ABSTRACT FROM AUTHOR]

Gingival fibroblasts (GFs) are abundant structural cells of the periodontium that contribute to the host's innate immunity by producing cytokines and chemokines in response to oral pathogens, such as Porphyromonas gingivalis. Isolated lipopolysaccharide (Pg‐LPS) is commonly used to study GF responses to P. gingivalis; however, this approach produced conflicting observations regarding its proinflammatory potential and the engagement of specific Toll‐like receptors (TLRs). In this work, we demonstrate that commercially available Pg‐LPS preparations are weak activators of GF innate immune responses compared with live P. gingivalis or other relevant virulence factors, such as P. gingivalis fimbriae or LPS from Escherichia coli. GF's nonresponsiveness to Pg‐LPS can be only partly attributed to the low expression of TLR4 and its accessory molecules, CD14 and LY36, and is likely caused by the unique structure and composition of the Pg‐LPS lipid A. Finally, we combined gene silencing and neutralizing antibody studies to demonstrate that GF response to infection with live P. gingivalis relies predominantly on TLR2. In contrast, the LPS‐TLR4 signaling plays a negligible role in inflammatory cytokine production by GFs exposed to this oral pathogen, confirming that Pg‐LPS stimulation is not an optimal model for studies of GF responses to P. gingivalis. [ABSTRACT FROM AUTHOR]

Background: Chronic periodontitis (CP), the most prevalent dysbiotic bacteria‐driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth‐surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. Methods: We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono‐ and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. Results: Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. Conclusions: We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency. [ABSTRACT FROM AUTHOR]

Periodontitis is a widespread chronic inflammatory disease caused by a changed dysbiotic oral microbiome. Although multiple species and risk factors are associated with periodontitis, Porphyromonas gingivalis has been identified as a keystone pathogen. The immune-modulatory function of P. gingivalis is well characterized, but the mechanism by which this bacterium secretes peptidyl arginine deiminase (PPAD), a protein/peptide citrullinating enzyme, thus contributing to the infinite feed-forward loop of inflammation, is not fully understood. To determine the functional role of citrullination in periodontitis, neutrophils were stimulated by P. gingivalis bearing wild-type PPAD and by a PPAD mutant strain lacking an active enzyme. Flow cytometry showed that PPAD contributed to prolonged neutrophil survival upon bacterial stimulation, accompanied by the secretion of aberrant IL-6 and TNF-α. To further assess the complex mechanism by which citrullination sustains a chronic inflammatory state, the ROS production and phagocytic activity of neutrophils were evaluated. Flow cytometry and colony formation assays showed that PPAD obstructs the resolution of inflammation by promoting neutrophil survival and the release of pro-inflammatory cytokines, while enhancing the resilience of the bacteria to phagocytosis. [ABSTRACT FROM AUTHOR]

Bacterial fibrillar adhesins are specialized extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin‐mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn)‐binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonization by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X‐ray crystal structure of the CshB adhesive domain NR2 and characterizing its Fn‐binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7‐fold reduction in Fn‐binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn‐binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram‐positive and Gram‐negative bacteria, where they are found housed within functionally cryptic multi‐domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined polymer adhesin domain (PAD) family of bacterial proteins. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is an oral pathogen that promotes dysbiosis by quenching the bactericidal activity of the host immune system while maintaining chronic inflammation, leading to periodontitis. This involves the secretion of virulence factors such as P. gingivalis peptidyl arginine deiminase (PPAD), which converts the C‐terminal Arg residues of bacterial and host‐derived proteins and peptides into citrulline. We have previously shown that PPAD activity and major fimbriae (containing FimA) are necessary for P. gingivalis to activate Toll‐like receptor 2 (TLR2). TLR2 is an important component of the innate immune system and plays a predominant role in the recognition of P. gingivalis by host cells. Here, we extend those findings to show that P. gingivalis strains deficient for PPAD and fimbriae induced almost identical transcriptional profiles in infected primary human gingival fibroblasts (PHGFs), but these differed substantially from the transcriptome elicited by the wild‐type ATCC 33277 strain. Apparently, PPAD‐modified fimbriae trigger the host cell response to P. gingivalis, as confirmed by showing that the proinflammatory host cell response mediated by TLR2 is dependent on PPAD activity and the presence of fimbriae, with type I fimbriae as the most potent TLR2 activators. We also found that PPAD‐modified accessory fimbrial subunits (FimC, FimD, and FimE) alone or in combination are TLR2 ligands in a reporter cell line. Although FimA polymerization to form the fimbrial shaft was not required for TLR2 activation, the secretion and proteolytic maturation of FimA were necessary for signaling by accessory Fim proteins. This was supported by showing that the proinflammatory activation of PHGFs is dependent on PPAD and accessory fimbrial subunits. We conclude that accessory fimbrial subunits are modified by PPAD and stimulate the response to P. gingivalis infection in a TLR2‐dependent manner. [ABSTRACT FROM AUTHOR]

Background: Periodontitis and oral pathogenic bacteria can contribute to the development of rheumatoid arthritis (RA). A connection between serum antibodies to Porphyromonas gingivalis (P. gingivalis) and RA has been established, but data on saliva antibodies to P. gingivalis in RA are lacking. We evaluated antibodies to P. gingivalis in serum and saliva in two Swedish RA studies as well as their association with RA, periodontitis, antibodies to citrullinated proteins (ACPA), and RA disease activity. Methods: The SARA (secretory antibodies in RA) study includes 196 patients with RA and 101 healthy controls. The Karlskrona RA study includes 132 patients with RA ≥ 61 years of age, who underwent dental examination. Serum Immunoglobulin G (IgG) and Immunoglobulin A (IgA) antibodies and saliva IgA antibodies to the P. gingivalis-specific Arg-specific gingipain B (RgpB) were measured in patients with RA and controls. Results: The level of saliva IgA anti-RgpB antibodies was significantly higher among patients with RA than among healthy controls in multivariate analysis adjusted for age, gender, smoking, and IgG ACPA (p = 0.022). Saliva IgA anti-RgpB antibodies were associated with RA disease activity in multivariate analysis (p = 0.036). Anti-RgpB antibodies were not associated with periodontitis or serum IgG ACPA. Conclusion: Patients with RA had higher levels of saliva IgA anti-RgpB antibodies than healthy controls. Saliva IgA anti-RgpB antibodies may be associated with RAdisease activity but were not associated with periodontitis or serum IgG ACPA. Our results indicate a local production of IgA anti-RgpB in the salivary glands that is not accompanied by systemic antibody production. [ABSTRACT FROM AUTHOR]

The Msp protein complex and the serine protease dentilisin are the best‐characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full‐sized dentipain (74‐kDa) and the 52‐kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin‐ and the Msp‐deficient mutants. Furthermore, dentipain was barely detected in the wild‐type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum‐free medium than in serum‐containing medium. Several genes, including those encoding transporters and methyl‐accepting chemotaxis proteins, were differentially expressed in the dentipain‐deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild‐type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum‐free medium than the wild‐type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum‐rich conditions. [ABSTRACT FROM AUTHOR]

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways. [ABSTRACT FROM AUTHOR]