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Increasing evidence has shown that a major periodontal pathobiont, Porphyromonas gingivalis , triggers oral dysbiosis leading to deterioration not only of periodontal health, but also of several systemic conditions. In the present study we identified remarkable anti- P. gingivalis activity of Foeniculum vulgare (fennel), an herbal plant used in Asian cuisine as well as in traditional medicine, by screening of 92 extracts prepared from 23 edible plants. The n-hexane-extracted fennel (HEF) showed a rapid lethal action toward P. gingivalis , while it was rather ineffective with a wide range of other oral commensal bacterial species. Morphological analysis using both high-speed atomic force microscopy and field emission scanning electron microscopy revealed that a low concentration of HEF (8 μg/mL) resulted in formation of protruding nanostructures composed of outer membrane vesicle (OMV)-like particles, while a high concentration of HEF (64 μg/mL) induced bacteriolysis with overproduction of OMVs with unusual surface properties. Interestingly, HEF treatment resulted in deprivation of two outer membrane transporter proteins, RagA and RagB, which is essential for nutrient acquisition in P. gingivalis , by extracellularly releasing RagA/RagB-enriched OMVs. Furthermore, HEF showed gingipain-inhibitory activity toward both arginine-specific (Rgps) and lysine-specific (Kgp) gingipains, resulting in blocking oral epithelial cell rounding and the subsequent detachment from culture dishes. Finally, we isolated petroselinic acid as a major bactericide as well as a gingipain inhibitor through a bioassay-guided fractionation of HEF. Taken together, our findings suggest clinical applicability of HEF and petroselinic acid for periodontitis therapy to eliminate P. gingivalis and its major virulence factors on the basis of the dual anti- P. gingivalis activity, i.e., rapid bacteriolysis and gingipain inhibition. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is an opportunistic Gram-positive bacterial pathogen that causes a wide variety of infectious diseases, including S. aureus bacteremia (SAB). Recent studies showed that rheumatoid arthritis (RA) is a risk factor for SAB, as RA patients appear to be more susceptible to SAB and display higher degrees of disease severity or complications, such as osteoarticular infections. On the other hand, Porphyromonas gingivalis is a Gram-negative bacterial oral pathogen, which is notable for its implication in the etiopathogenesis of RA due to its unique citrullinating enzyme PPAD and its highly effective proteases, known as gingipains. Both PPAD and gingipains are abundant in P. gingivalis outer membrane vesicles (OMVs), which are secreted nanostructures that originate from the outer membrane. Here we show that P. gingivalis OMVs cause the aggregation of S. aureus bacteria in a gingipain- and PPAD-dependent fashion, and that this aggregation phenotype is reversible. Importantly, we also show that the exposure of S. aureus to OMVs of P. gingivalis promotes the staphylococcal internalization by human neutrophils with no detectable neutrophil killing. Altogether, our observations suggest that P. gingivalis can eliminate its potential competitor S. aureus by promoting staphylococcal aggregation and the subsequent internalization by neutrophils. We hypothesize that this phenomenon may have repercussions for the host, since immune cells with internalized bacteria may facilitate bacterial translocation to the blood stream, which could potentially contribute to the association between RA and SAB.

Increasing evidence has shown that a major periodontal pathobiont, Porphyromonas gingivalis, triggers oral dysbiosis leading to deterioration not only of periodontal health, but also of several systemic conditions. In the present study we identified remarkable anti-P. gingivalis activity of Foeniculum vulgare (fennel), an herbal plant used in Asian cuisine as well as in traditional medicine, by screening of 92 extracts prepared from 23 edible plants. The n-hexane-extracted fennel (HEF) showed a rapid lethal action toward P. gingivalis, while it was rather ineffective with a wide range of other oral commensal bacterial species. Morphological analysis using both high-speed atomic force microscopy and field emission scanning electron microscopy revealed that a low concentration of HEF (8 μg/mL) resulted in formation of protruding nanostructures composed of outer membrane vesicle (OMV)-like particles, while a high concentration of HEF (64 μg/mL) induced bacteriolysis with overproduction of OMVs with unusual surface properties. Interestingly, HEF treatment resulted in deprivation of two outer membrane transporter proteins, RagA and RagB, which is essential for nutrient acquisition in P. gingivalis, by extracellularly releasing RagA/RagB-enriched OMVs. Furthermore, HEF showed gingipain-inhibitory activity toward both arginine-specific (Rgps) and lysine-specific (Kgp) gingipains, resulting in blocking oral epithelial cell rounding and the subsequent detachment from culture dishes. Finally, we isolated petroselinic acid as a major bactericide as well as a gingipain inhibitor through a bioassay-guided fractionation of HEF. Taken together, our findings suggest clinical applicability of HEF and petroselinic acid for periodontitis therapy to eliminate P. gingivalis and its major virulence factors on the basis of the dual anti-P. gingivalis activity, i.e., rapid bacteriolysis and gingipain inhibition.

Periodontal diseases initiate on epithelial surfaces of the subgingival compartment, while the gingival epithelium functions as an epithelial barrier against microbial infection and orchestrates immune responses. Porphyromonas gingivalis is a major pathogen of periodontal diseases and has an ability to penetrate the epithelial barrier. To assess the molecular basis of gingival epithelial barrier dysfunction associated with P. gingivalis, we newly developed a three-dimensional multilayered tissue model of gingival epithelium with gene manipulation. Using this novel approach, P. gingivalis gingipains including Arg- or Lys-specific cysteine proteases were found to specifically degrade junctional adhesion molecule 1 and coxsackievirus and adenovirus receptor in the tissue model, leading to increased permeability for lipopolysaccharide, peptidoglycan, and gingipains. This review summarizes the strategy used by P. gingivalis to disable the epithelial barrier by disrupting specific junctional adhesion molecules.

"Chronic" periodontitis and its keystone pathogen Porphyromonas gingivalis have repeatedly been associated with Alzheimer's disease (AD). Pathological hallmarks in AD are brain accumulations of amyloid-beta and neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. In addition, neuroinflammation induced by P. gingivalis has increasingly been recognized as a factor in the pathogenesis of AD. The present mini-review discusses possible mechanisms for the induction of neuroinflammation by P. gingivalis in AD, involving factors such as pro-inflammatory mediators, amyloid-beta, tau, microglia, cathepsin B, and protein kinase R. Inflammagens of P. gingivalis such as lipopolysaccharide and gingipains are also discussed. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, a bacterium associated with periodontal disease, is a suspected cause of Alzheimer’s disease. This bacterium is reliant on gingipain proteases, which cleave host proteins after arginine and lysine residues. To characterize gingipain susceptibility, we performed enrichment analyses of arginine and lysine proportion proteome-wide. Genes differentially expressed in brain samples with detected P. gingivalis reads were also examined. Genes from these analyses were tested for functional enrichment and specific neuroanatomical expression patterns. Proteins in the SRP-dependent cotranslational protein targeting to membrane pathway were enriched for these residues and previously associated with periodontal and Alzheimer’s disease. These ribosomal genes are up-regulated in prefrontal cortex samples with detected P. gingivalis sequences. Other differentially expressed genes have been previously associated with dementia (ITM2B, MAPT, ZNF267, and DHX37). For an anatomical perspective, we characterized the expression of the P. gingivalis associated genes in the mouse and human brain. This analysis highlighted the hypothalamus, cholinergic neurons, and the basal forebrain. Our results suggest markers of neural P. gingivalis infection and link the cholinergic and gingipain hypotheses of Alzheimer’s disease.

Porphyromonas gingivalis , a bacterium associated with periodontal disease, is a suspected cause of Alzheimer's disease. This bacterium is reliant on gingipain proteases, which cleave host proteins after arginine and lysine residues. To characterize gingipain susceptibility, we performed enrichment analyses of arginine and lysine proportion proteome-wide. Genes differentially expressed in brain samples with detected P. gingivalis reads were also examined. Genes from these analyses were tested for functional enrichment and specific neuroanatomical expression patterns. Proteins in the SRP-dependent cotranslational protein targeting to membrane pathway were enriched for these residues and previously associated with periodontal and Alzheimer's disease. These ribosomal genes are up-regulated in prefrontal cortex samples with detected P. gingivalis sequences. Other differentially expressed genes have been previously associated with dementia (ITM2B , MAPT , ZNF267 , and DHX37). For an anatomical perspective, we characterized the expression of the P. gingivalis associated genes in the mouse and human brain. This analysis highlighted the hypothalamus, cholinergic neurons, and the basal forebrain. Our results suggest markers of neural P. gingivalis infection and link the cholinergic and gingipain hypotheses of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

As one of the main pathogens of periodontitis, Porphyromonas gingivalis often forms mixed biofilms with other bacteria or fungi under the gingiva, such as Candida albicans. Heme is an important iron source for P. gingivalis and C. albicans that supports their growth in the host. From the perspective of heme competition, this study aims to clarify that the competition for heme enhances the pathogenic potential of P. gingivalis during the interaction between P. gingivalis and C. albicans. Porphyromonas gingivalis single-species biofilm and P. gingivalis-C. albicans dual-species biofilm were established in a low- and high-heme environment. The results showed that the vitality of P. gingivalis was increased in the dual-species biofilm under the condition of low heme, and the same trend was observed under a laser confocal microscope. Furthermore, the morphological changes in P. gingivalis were observed by electron microscope, and the resistance of P. gingivalis in dual-species biofilm was stronger against the killing effect of healthy human serum and antibiotics. The ability of P. gingivalis to agglutinate erythrocyte was also enhanced in dual-species biofilm. These changes disappeared when heme was sufficient, which confirmed that heme competition was the cause of thepathogenicy change in P. gingivalis. Gene level analysis showed that P. gingivalis was in a superior position in the competition relationship by increasing the expression of heme utilization-related genes, such as HmuY, HmuR, HusA, and Tlr. In addition, the expression of genes encoding gingipains (Kgp, RgpA/B) was also significantly increased. They not only participate in the process of utilizing heme, but also are important components of the virulence factors of P. gingivalis. In conclusion, our results indicated that the pathogenic potential of P. gingivalis was enhanced by C. albicans through heme competition, which ultimately promoted the occurrence and development of periodontitis and, therefore, C. albicans subgingival colonization should be considered as a factor in assessing the risk of periodontitis.

Porphyromonas gingivalis, the main etiologic agent of periodontitis, secretes cysteine proteases named gingipains. HRgpA and RgpB gingipains have Arg-specificity, while Kgp gingipain is Lys-specific. Together they can cleave an array of proteins and importantly contribute to the development of periodontitis. In this study we focused on gingipain-exerted proteolysis at the cell surface of human gingival epithelial cells [telomerase immortalized gingival keratinocytes (TIGK)] in order to better understand the molecular mechanisms behind tissue destruction in periodontitis. Using mass spectrometry, we investigated the whole sheddome/degradome of TIGK cell surface proteins by P. gingivalis strains differing in gingipain expression and by purified gingipains, and performed the first global proteomic analysis of gignpain proteolysis at the membrane. Incubation of TIGK cells with P. gingivalis resulted in massive degradation of proteins already at low multiplicity of infection, whereas incubating cells with purified gingipains resulted in more discrete patterns, indicative of a combination of complete degradation and shedding of membrane proteins. Most of the identified gingipain substrates were molecules involved in adhesion, suggesting that gingipains may cause tissue damage through cleavage of cell contacts, resulting in cell detachment and rounding, and consequently leading to anoikis. However, HRgpA and RgpB gingipains differ in their mechanism of action. While RgpB rapidly degraded the proteins, HRgpA exhibited a much slower proteolysis indicative of ectodomain shedding, as demonstrated for the transferrin receptor protein 1 (TFRC). These results reveal a molecular underpinning to P. gingivalis-induced tissue destruction and enhance our knowledge of the role of P. gingivalis proteases in the pathobiology of periodontitis. Proteomics data are available via ProteomeXchange with identifier PXD015679.

As one of the main pathogens of periodontitis, Porphyromonas gingivalis often forms mixed biofilms with other bacteria or fungi under the gingiva, such as Candida albicans. Heme is an important iron source for P. gingivalis and C. albicans that supports their growth in the host. From the perspective of heme competition, this study aims to clarify that the competition for heme enhances the pathogenic potential of P. gingivalis during the interaction between P. gingivalis and C. albicans. Porphyromonas gingivalis single-species biofilm and P. gingivalis - C. albicans dual-species biofilm were established in a low- and high-heme environment. The results showed that the vitality of P. gingivalis was increased in the dual-species biofilm under the condition of low heme, and the same trend was observed under a laser confocal microscope. Furthermore, the morphological changes in P. gingivalis were observed by electron microscope, and the resistance of P. gingivalis in dual-species biofilm was stronger against the killing effect of healthy human serum and antibiotics. The ability of P. gingivalis to agglutinate erythrocyte was also enhanced in dual-species biofilm. These changes disappeared when heme was sufficient, which confirmed that heme competition was the cause of thepathogenicy change in P. gingivalis. Gene level analysis showed that P. gingivalis was in a superior position in the competition relationship by increasing the expression of heme utilization-related genes, such as HmuY, HmuR, HusA, and Tlr. In addition, the expression of genes encoding gingipains (Kgp, RgpA/B) was also significantly increased. They not only participate in the process of utilizing heme, but also are important components of the virulence factors of P. gingivalis. In conclusion, our results indicated that the pathogenic potential of P. gingivalis was enhanced by C. albicans through heme competition, which ultimately promoted the occurrence and development of periodontitis and, therefore, C. albicans subgingival colonization should be considered as a factor in assessing the risk of periodontitis. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis , the main etiologic agent of periodontitis, secretes cysteine proteases named gingipains. HRgpA and RgpB gingipains have Arg-specificity, while Kgp gingipain is Lys-specific. Together they can cleave an array of proteins and importantly contribute to the development of periodontitis. In this study we focused on gingipain-exerted proteolysis at the cell surface of human gingival epithelial cells [telomerase immortalized gingival keratinocytes (TIGK)] in order to better understand the molecular mechanisms behind tissue destruction in periodontitis. Using mass spectrometry, we investigated the whole sheddome/degradome of TIGK cell surface proteins by P. gingivalis strains differing in gingipain expression and by purified gingipains, and performed the first global proteomic analysis of gignpain proteolysis at the membrane. Incubation of TIGK cells with P. gingivalis resulted in massive degradation of proteins already at low multiplicity of infection, whereas incubating cells with purified gingipains resulted in more discrete patterns, indicative of a combination of complete degradation and shedding of membrane proteins. Most of the identified gingipain substrates were molecules involved in adhesion, suggesting that gingipains may cause tissue damage through cleavage of cell contacts, resulting in cell detachment and rounding, and consequently leading to anoikis. However, HRgpA and RgpB gingipains differ in their mechanism of action. While RgpB rapidly degraded the proteins, HRgpA exhibited a much slower proteolysis indicative of ectodomain shedding, as demonstrated for the transferrin receptor protein 1 (TFRC). These results reveal a molecular underpinning to P. gingivalis -induced tissue destruction and enhance our knowledge of the role of P. gingivalis proteases in the pathobiology of periodontitis. Proteomics data are available via ProteomeXchange with identifier PXD015679. [ABSTRACT FROM AUTHOR]

Parkinson’s disease (PD) is a well-known neurodegenerative disease with a strong association established with systemic inflammation. Recently, the role of the gingipain protease group from Porphyromonas gingivalis was implicated in Alzheimer’s disease and here we present evidence, using a fluorescent antibody to detect gingipain R1 (RgpA), of its presence in a PD population. To further elucidate the action of this gingipain, as well as the action of the lipopolysaccharide (LPS) from P. gingivalis, low concentrations of recombinant RgpA and LPS were added to purified fluorescent fibrinogen. We also substantiate previous findings regarding PD by emphasizing the presence of systemic inflammation via multiplex cytokine analysis, and demonstrate hypercoagulation using thromboelastography (TEG), confocal and electron microscopy. Biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. In our PD and control blood analysis, our results show increased hypercoagulation, the presence of amyloid formation in plasma, and profound ultrastructural changes to platelets. Our laboratory analysis of purified fibrinogen with added RgpA, and/or LPS, showed preliminary data with regards to the actions of the protease and the bacterial membrane inflammagen on plasma proteins, to better understand the nature of established PD.

Periodontitis is a common intraoral infection and is inextricably linked to systemic diseases. Recently, the regulation between host immunologic response and periodontal pathogens has become a hotspot to explain the mechanism of periodontitis and related systemic diseases. Since Porphyromonas gingivalis (P. gingivalis) was proved as critical periodontal pathogen above all, researches focusing on the mechanism of its virulence factors have received extensive attention. Studies have shown that in the development of periodontitis, in addition to the direct release of virulent factors by periodontal pathogens to destroy periodontal tissues, over-low or over-high intrinsic immune and inflammatory response mediated by Toll-like receptors (TLRs) can lead to more lasting destruction of periodontal tissues. It is very necessary to sort out how various cytopathic factors of P. gingivalis mediate inflammation and immune responses between the host through TLRs so as to help precisely prevent, diagnose, and treat periodontitis in clinic. This review summarizes the role of three most widely studied pathogenic factors produced by P. gingivalis (lipopolysaccharide, gingipains, pili) and their interactions with TLRs at the cellular and molecular level in the progress of periodontitis.

Pancreatic cancer is a highly lethal disease, and most patients remain asymptomatic until the disease enters advanced stages. There is lack of knowledge in the pathogenesis, effective prevention and early diagnosis of pancreatic cancer. Recently, bacteria were found in pancreatic tissue that has been considered sterile before. The distribution of flora in pancreatic cancer tissue was reported to be different from normal pancreatic tissue. These abnormally distributed bacteria may be the risk factors for inducing pancreatic cancer. Therefore, studies on combined effect of multi-bacterial and multi-virulence factors may add to the knowledge of pancreatic cancer pathogenesis and aid in designing new preventive and therapeutic strategies. In this review, we outlined three oral bacteria associated with pancreatic cancer and their virulence factors linked with cancer. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a well-known neurodegenerative disease with a strong association established with systemic inflammation. Recently, the role of the gingipain protease group from Porphyromonas gingivalis was implicated in Alzheimer's disease and here we present evidence, using a fluorescent antibody to detect gingipain R1 (RgpA), of its presence in a PD population. To further elucidate the action of this gingipain, as well as the action of the lipopolysaccharide (LPS) from P. gingivalis , low concentrations of recombinant RgpA and LPS were added to purified fluorescent fibrinogen. We also substantiate previous findings regarding PD by emphasizing the presence of systemic inflammation via multiplex cytokine analysis, and demonstrate hypercoagulation using thromboelastography (TEG), confocal and electron microscopy. Biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. In our PD and control blood analysis, our results show increased hypercoagulation, the presence of amyloid formation in plasma, and profound ultrastructural changes to platelets. Our laboratory analysis of purified fibrinogen with added RgpA, and/or LPS, showed preliminary data with regards to the actions of the protease and the bacterial membrane inflammagen on plasma proteins, to better understand the nature of established PD. [ABSTRACT FROM AUTHOR]

Periodontitis is a common intraoral infection and is inextricably linked to systemic diseases. Recently, the regulation between host immunologic response and periodontal pathogens has become a hotspot to explain the mechanism of periodontitis and related systemic diseases. Since Porphyromonas gingivalis (P. gingivalis) was proved as critical periodontal pathogen above all, researches focusing on the mechanism of its virulence factors have received extensive attention. Studies have shown that in the development of periodontitis, in addition to the direct release of virulent factors by periodontal pathogens to destroy periodontal tissues, over-low or over-high intrinsic immune and inflammatory response mediated by Toll-like receptors (TLRs) can lead to more lasting destruction of periodontal tissues. It is very necessary to sort out how various cytopathic factors of P. gingivalis mediate inflammation and immune responses between the host through TLRs so as to help precisely prevent, diagnose, and treat periodontitis in clinic. This review summarizes the role of three most widely studied pathogenic factors produced by P. gingivalis (lipopolysaccharide, gingipains, pili) and their interactions with TLRs at the cellular and molecular level in the progress of periodontitis. [ABSTRACT FROM AUTHOR]

Gingipain cysteine proteases are considered key virulence factors of Porphyromonas gingivalis. They significantly influence antibacterial and homeostatic functions of macrophages, neutrophils, the complement system, and cytokine networks. Recent data indicate the role of P. gingivalis in T cell differentiation; however, the involvement of gingipains in this process remains elusive. Therefore, the aim of this study was to investigate the contribution of danger signals triggered by the gingipains on the generation of Th17 cells, which play a key role in protection against bacterial diseases but may cause chronic inflammation and bone resorption. To this end we compared the effects of the wild-type strain of P. gingivalis (W83) with its isogenic mutant devoid of gingipain activity (ΔKΔRAB), and bacterial cells pretreated with a highly-specific inhibitor of gingipains activity (KYTs). Antigen presenting cells (APCs), both professional (dendritic cells), and non-professional (gingival keratinocytes), exposed to viable bacteria expressed high amounts of cytokines (IL-6, IL-21, IL-23). These cytokines are reported to either stimulate or balance the Th17-dependent immune response. Surprisingly, cells infected with P. gingivalis devoid of gingipain activity showed increased levels of all tested cytokines compared to bacteria with fully active enzymes. The effect was dependent on both the reduction of cytokine proteolysis and the lack of cross-talk with other bacterial virulence factors, including LPS and fimbriae that induce de novo synthesis of cytokines. The profile of lymphocyte T differentiation from naive T cells showed enhanced generation of Th17 in response to bacteria with inactive gingipains. Moreover, we found that gingipain-dependent induction of Th17 cells was highly specific, since other T cell-subsets remained unchanged. Finally, inhibition of IL-6 signaling in dendritic cells led to a significant depletion of the Th17 population. Cumulatively, this study revealed a previously undisclosed role of gingipain activity in the process of Th17 differentiation reliant on blocking signaling through IL-6. Since inactivation of gingipains accelerates the skewing of T cells toward Th17 cells, which are detrimental in periodontitis, IL-6 signaling may serve as an attractive target for treatment of the disease.