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Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth‐essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability. [ABSTRACT FROM AUTHOR]

Background/purpose: Periodontitis is associated with various systemic diseases, potentially facilitated by the passage of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs). Several recent studies have suggested a connection between Pg-OMVs and neuroinflammation and neurodegeneration, but the precise causal relationship remains unclear. This study aimed to investigate the mechanisms underlying these associations using in vitro models. Materials and methods: Isolated Pg-OMVs were characterized by morphology, size, and gingipain activity. We exposed SH-SY5Y neuroblastoma cells and BV-2 microglial cells to various concentrations of Pg-OMVs. Cell morphology, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, an enzyme-linked immunosorbent assay, and Western blot analysis were used to evaluate the cellular mechanism underlying Pg-OMV-induced neurotoxicity in neuronal cells and inflammatory responses in microglial cells. Results: Exposure to Pg-OMVs induced neurotoxicity in SH-SY5Y cells, as evidenced by cellular shrinkage, reduced viability, activation of apoptotic pathways, and diminished neuronal differentiation markers. Gingipain inhibition mitigated these effects, suggesting that gingipain mediates Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. Our research on neuroinflammation suggests that upon endocytosis of Pg-OMVs by BV-2 cells, lipopolysaccharide (LPS) can modulate the production of inducible nitric oxide synthase and tumor necrosis factor-alpha by activating pathways that involve phosphorylated AKT and the phosphorylated JNK pathway. Conclusion: Our study demonstrated that following the endocytosis of Pg-OMVs, gingipain can induce neurotoxicity in SH-SY5Y cells. Furthermore, the Pg-OMVs-associated LPS can trigger neuroinflammation via AKT and JNK signaling pathways in BV-2 cells.

Purpose: To evaluate the inhibitory effects of Streptococcus salivarius K12 and M18 strains on the growth of six oral pathogens as well as their release of volatile sulfur compounds (VSCs), and whether these probiotics can inhibit the expression of arginine-specific gingipain A (RgpA), a protease secreted by Porphyromonas gingivalis. Materials and Methods: After six halitogenic oral pathogens (P. gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, and Eikenella corrodens) were cultured with or without S. salivarius K12 and M18, the concentration of two VSCs was measured. Moreover, the antimicrobial activity of S. salivarius K12 and M18 against these pathogens and the suppressive effect on RgpA release by P. gingivalis were assessed. Results: In the co-culture of S. salivarius K12 or M18 with oral pathogenic bacteria, the growth of all six oral pathogens was significantly inhibited (p < 0.01). Additionally, S. salivarius K12 and M18 had an inhibitory effect on the production of the halitogenic substances H2S and CH3SH (p < 0.01) as well as the expression of P. gingivalis RgpA. Finally, we demonstrated that the addition of only culture supernatants of the two strains K12/M18 to oral pathogen cultures was sufficient to mimic the effects of K12/M18 co-cultures upon VSCs production and protease expression. Conclusions: S. salivarius K12 and M18 inhibited VSC release by all six of the major oral pathogens that were assayed and reduced the expression of RgpA. [ABSTRACT FROM AUTHOR]

Periodontitis is associated with various systemic diseases, potentially facilitated by the passage of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs). Several recent studies have suggested a connection between Pg-OMVs and neuroinflammation and neurodegeneration, but the precise causal relationship remains unclear. This study aimed to investigate the mechanisms underlying these associations using in vitro models. Isolated Pg-OMVs were characterized by morphology, size, and gingipain activity. We exposed SH-SY5Y neuroblastoma cells and BV-2 microglial cells to various concentrations of Pg-OMVs. Cell morphology, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, an enzyme-linked immunosorbent assay, and Western blot analysis were used to evaluate the cellular mechanism underlying Pg-OMV-induced neurotoxicity in neuronal cells and inflammatory responses in microglial cells. Exposure to Pg-OMVs induced neurotoxicity in SH-SY5Y cells, as evidenced by cellular shrinkage, reduced viability, activation of apoptotic pathways, and diminished neuronal differentiation markers. Gingipain inhibition mitigated these effects, suggesting that gingipain mediates Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. Our research on neuroinflammation suggests that upon endocytosis of Pg-OMVs by BV-2 cells, lipopolysaccharide (LPS) can modulate the production of inducible nitric oxide synthase and tumor necrosis factor-alpha by activating pathways that involve phosphorylated AKT and the phosphorylated JNK pathway. Our study demonstrated that following the endocytosis of Pg-OMVs, gingipain can induce neurotoxicity in SH-SY5Y cells. Furthermore, the Pg-OMVs-associated LPS can trigger neuroinflammation via AKT and JNK signaling pathways in BV-2 cells. [ABSTRACT FROM AUTHOR]

Our objectives of this narrative review is to summarize the scientific perspective on the effect of periodontal disease on age-related diseases, focusing on chronic low-level systemic inflammation, and discuss the goals for future research. In this review, we summarized the findings of articles that showed the mechanisms of periodontal disease and each age-related disease. Periodontal disease is a prevalent infectious disease due to oral anaerobic bacteria that results in dissolution of the gums around the teeth (gingiva) and the bone supporting the teeth, leading to decreased quality of life. The disease is more prevalent with increasing age and associated with age-related diseases, such as diabetes, non-alcoholic fatty liver disease, cardiovascular disease, dementia, and osteoporosis via chronic low-level systemic inflammation, which requires long-term care and increases medical costs. Chronic low-level systemic inflammation due to periodontal disease is a common risk factor for age-related diseases, but the mechanisms of inflammation in each disease vary. Periodontal pathogens mainly cause three patterns of inflammation: cytokine crosstalk that attacks the whole body, changes in the balance of gut microbiota, and production of proteases like gingipains that can subvert immune responses to reach various systemic organs, especially the brain. Several age-related diseases, such as diabetes and dementia, can be improved by treating periodontal disease as inflammatory markers are reduced. In conclusion, it will be necessary to confirm whether the exact mechanism in the human body is the same as that in mice. Improvement in chronic low-level systemic inflammation caused by periodontal disease may be effective in the treatment of age-related diseases.

Objective·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6.Methods·The HN6 cell line was selected, cultivated, and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis: control group, 3.125 μg/mL group, 6.25 μg/mL group, 12.5 μg/mL group, 25 μg/mL group, 50 μg/mL group, and 100 μg/mL group. After 24 and 48 h of cultivation, CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity. Subsequent experiments were divided into control group, 25 μg/mL group and 50 μg/mL group. Flow cytometry was used to examine the effects of gingipain extract on cell cycle. Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability. Real-time PCR (RT-PCR) and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells.Results·Stimulated with gingipain extract for 24 h, the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL (P=0.025), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.049) groups compared to the control group. After 48 h, proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024), 12.5 μg/mL (P=0.006), 25 μg/mL (P=0.000), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.000) groups compared to the control group. Cell cycle analysis revealed that, after 24 h of gingipain stimulation, the proportion of HN6 cells in the G1 phase decreased, while the proportion in the S+G2 phase significantly increased compared to the control group (25 μg/mL group: P=0.024; 50 μg/mL group: P=0.001). Compared to the control group, the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased (P=0.001). Compared to the control group, the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased. RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased, the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased, while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group.Conclusion·Gingipain extract could promote proliferation, migration, and invasion of oral squamous cell carcinoma HN6 cells.

Objective. To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6. Methods. The HN6 cell line was selected, cultivated, and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis: control group, 3.125 µg/mL. group, 6.25 µg/mL. group, 12.5 µg/mL group, 25 µg/mL group, 50 µg/mL. group, and 100 µg/ml. group. After 24 and 48 h of cultivation, CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity. Subsequent experiments were divided into control group, 25 µg/mL. group and 50 µg/mL group. Flow cytometry was used to examine the effects of gingipain extract on cell cycle. Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability. Real-time PCR (RT-PCR) and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells. Results Stimulated with gingipain extract for 24 h, the HN6 cells showed significantly increased proliferation activity in the 25 µg/mL. (P-0.025), 50 µg/mL (P-0.000), and 100 µg/mL (P-0.049) groups compared to the control group. After 48 h, proliferation activity was significantly higher in the 6.25 µg/mL (P-0.024), 12.5 µg/mL (P-0.006), 25 µg/mL (P-0.000), 50 µg/mL (P-0.000), and 100 µg/mL (P-0.000) groups compared to the control group. Cell cycle analysis revealed that, after 24 h of gingipain stimulation, the proportion of HN6 cells in the G1 phase decreased, while the proportion in the S+G2 phase significantly increased compared to the control group (25 µg/mL. group: P-0.024; 50 µg/mL. group: P-0.001). Compared to the control group, the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased (P-0.001). Compared to the control group, the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased. RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased, the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased, while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group. Conclusion Gingipain extract could promote proliferation, migration, and invasion of oral squamous cell carcinoma HN6 cells. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU mL-1 bacteria in vitro. RgpB activity was imaged in porcine jaws with low-nanomolar sensitivity. Diagnostic efficacy was evaluated in gingival crevicular fluid samples from subjects with and without periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson's r=0.71). Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, with relevance and potential utility for disease models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.

ABSTRACTPorphyromonas gingivalis strains exhibit different phenotypes in vitro, different virulence potential in animal models, and different associations with human diseases, with strains classified as virulent/more virulent (e.g., A7436 and W83) or as less virulent/avirulent (e.g., ATCC 33277). In this study, we comparatively analyzed the A7436 and ATCC 33277 strains to better understand their variability. Global gene expression analysis in response to heme and iron limitation revealed more pronounced differences in the A7436 than in the ATCC 33277 strain; however, in both strains, the largest changes were observed in genes encoding hypothetical proteins, genes whose products participate in energy metabolism, and in genes encoding proteins engaged in transport and binding proteins. Our results confirmed that variability between P. gingivalis strains is due to differences in the arrangement of their genomes. Analysis of gene expression of heme acquisition systems demonstrated that not only the availability of iron and heme in the external environment but also the ability to store iron intracellularly can influence the P. gingivalis phenotype. Therefore, we assume that differences in virulence potential may also be due to differences in the production of systems involved in iron and heme acquisition, mainly the Hmu system. In addition, our study showed that hemoglobin, in a concentration-dependent manner, differentially influences the virulence potential of P. gingivalis strains. We conclude that iron and heme homeostasis may add to the variability observed between P. gingivalis strains.IMPORTANCEPeriodontitis belongs to a group of multifactorial diseases, characterized by inflammation and destruction of tooth-supporting tissues. P. gingivalis is one of the most important microbial factors involved in the initiation and progression of periodontitis. To survive in the host, the bacterium must acquire heme as a source of iron and protoporphyrin IX. P. gingivalis strains respond differently to changing iron and heme concentrations, which may be due to differences in the expression of systems involved in iron and heme acquisition. The ability to accumulate iron intracellularly, being different in more and less virulent P. gingivalis strains, may influence their phenotypes, production of virulence factors (including proteins engaged in heme acquisition), and virulence potential of this bacterium.

Purpose : To assess the effects of orthodontic treatment on the changes of Porphyromonas gingivalis (P.g) and gingipains as well as gingival inflammation. Materials and methods : A total of 105 patients who had healthy gingivae and were treated as research subjects, the gingival crevicular fluid of whom was all collected before treatment and in the 1st, 2nd, 3rd, and 6th months after wearing an appliance. Then, the 16S ribosomal deoxyribonucleic acid (rDNA) polymerase chain reaction (PCR) technique was employed to detect the levels of P.g, Lys-gingipain (Kgp) and Arg-gingipain (Rgp) in the specimens. It was followed by a statistical analysis of the data via the SPSS 23.0 software package. Results : In P.g detection, the 16S rDNA PCR technique presented specific advantages and high sensitivity. The positive detection rate of P.g in the 1st, 2nd and 3rd month of treatment (p <.05), that of Kgp in P.g -positive patients in the 2nd and 3rd months of treatment (p <.05), and that of Rgp in P.g -positive patients in the 2nd and 3rd months of treatment (p <.05) were higher than the pre-treatment results. Compared with gingival index (GI)-0 patients, GI-1 patients exhibited higher positive detection rates of P.g and Kgp and Rgp, which were further found to be positively correlated with the treatment duration and GI (p <.05). Conclusion : In the early stage of orthodontic treatment, the levels of P.g, Kgp and Rgp rise after wearing the appliance, inducing gingival inflammation. [ABSTRACT FROM AUTHOR]

Objective: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of Alzheimer's disease (AD). Whether Pg or its contents can reach the brain and directly affect neuropathology is, however, unknown. Here, we investigated whether outer membrane vesicles (OMVs) of Pg translocate to the brain and induce the pathogenic features of AD. Material and Methods: Pg OMVs were injected into the abdominal cavity of mice for 12 weeks. Pg OMV translocation to the brain was detected by immunohistochemistry using an anti‐gingipain antibody. Tau protein and microglial activation in the mouse brain were examined by western blotting and immunohistochemistry. The effect of gingipains on inflammation was assessed by real‐time polymerase chain reaction using human microglial HMC3 cells. Results: Gingipains were detected in the region around cerebral ventricles, choroid plexus, and ventricular ependymal cells in Pg OMV‐administered mice. Tau and phosphorylated Tau protein increased and microglia were activated. Pg OMVs also increased the gene expression of proinflammatory cytokines in HMC3 cells in a gingipain‐dependent manner. Conclusion: Pg OMVs, including gingipains, can reach the cerebral ventricle and induce neuroinflammation by activating microglia. Pg OMVs may provide a better understanding of the implications of periodontal diseases in neurodegenerative conditions such as AD. [ABSTRACT FROM AUTHOR]

Objective: Porphyromonas gingivalis (P. gingivalis), a major periodontopathogen, is associated with overfeeding disorders, including metabolic syndrome. Gingipains are one of the most powerful endotoxins of P. gingivalis. Our aim was to reveal the effects of gingipain injections on the intestinal oxidant-antioxidant status and alkaline phosphatase (ALP) activity in overfed zebrafish. Materials and Methods: Four groups of healthy adult zebrafish were placed in random tanks as C: Control (n=15); GP: Gingipain (n=15); OF: Overfeeding (n=15); and OF+GP: Overfeeding+Gingipain (n=15) groups. At the end of the experiment, levels of intestinal lipid peroxidation (LPO) and ALP, glutathione S-transferase (GST), and catalase (CAT) activities were evaluated. Results: Intestinal LPO was significantly lower in the GP and OF groups compared to C. Gingipain injection in OF (OF+GP) significantly elevated LPO when compared to C, GP, and OF groups. ALP activities decreased significantly in the GP, OF, and OF+GP compared to C. GST activities increased significantly in the GP when compared to C. Decreased GST activities were observed in the OF and OF+GP. This decrease was less in OF+GP. CAT activities significantly decreased in all groups when compared to C. Conclusion: Our findings demonstrate that gingipain injection alters the ALP activity and intestinal oxidant-antioxidant status in overfed zebrafish. [ABSTRACT FROM AUTHOR]

Purpose: To assess the effects of orthodontic treatment on the changes of Porphyromonas gingivalis (P.g) and gingipains as well as gingival inflammation. Materials and methods: A total of 105 patients who had healthy gingivae and were treated as research subjects, the gingival crevicular fluid of whom was all collected before treatment and in the 1st, 2nd, 3rd, and 6th months after wearing an appliance. Then, the 16S ribosomal deoxyribonucleic acid (rDNA) polymerase chain reaction (PCR) technique was employed to detect the levels of P.g, Lys-gingipain (Kgp) and Arg-gingipain (Rgp) in the specimens. It was followed by a statistical analysis of the data via the SPSS 23.0 software package. Results: In P.g detection, the 16S rDNA PCR technique presented specific advantages and high sensitivity. The positive detection rate of P.g in the 1st, 2nd and 3rd month of treatment (p < .05), that of Kgp in P.g-positive patients in the 2nd and 3rd months of treatment (p < .05), and that of Rgp in P.g-positive patients in the 2nd and 3rd months of treatment (p < .05) were higher than the pre-treatment results. Compared with gingival index (GI)-0 patients, GI-1 patients exhibited higher positive detection rates of P.g and Kgp and Rgp, which were further found to be positively correlated with the treatment duration and GI (p < .05). Conclusion: In the early stage of orthodontic treatment, the levels of P.g, Kgp and Rgp rise after wearing the appliance, inducing gingival inflammation. [ABSTRACT FROM AUTHOR]

Influenza is a worldwide health problem that causes significant morbidity and mortality among the elderly; therefore, its prevention is important. During influenza virus infection, the cleavage of hemagglutinin (HA) is essential for the virus to enter host cells. Influenza virus-bacteria interactions influence the pathogenicity of infections, and specific bacteria contribute to the severity of the disease by participating in HA cleavage. Poor oral hygiene and the presence of oral bacteria are associated with influenza. Porphyromonas gingivalis, a periodontopathic bacterium, is particularly associated with influenza; however, the underlying mechanisms remain unclear. In the present study, we observed P. gingivalis culture supernatant promoted viral release and cell-to-cell spread of the infection. Further investigation revealed that the supernatant contained cleaved HA. Therefore, we focused on gingipains (Rgp and Kgp) which are trypsin-like proteases produced by P. gingivalis. We determined that the Rgp inhibitor inhibited both HA cleavage and the increase in virus release associated with the P. gingivalis culture supernatant, whereas such effects were not observed with the Kgp inhibitor. In addition, Rgp-deficient P. gingivalis culture supernatant failed to cleave HA, enhance virus spread, or increase virus release. In contrast, Kgp-deficient P. gingivalis culture supernatant cleaved HA and promoted infection. These results indicated that P. gingivalis-secreted Rgp has the potential to activate influenza virus infectivity through HA cleavage, suggesting that understanding the effects of P. gingivalis on influenza virus infection will contribute to the establishment of influenza prevention measures., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis expresses a broad array of virulence factors that enable it to play a central role in the etiopathogenesis of periodontitis. The objective of the present study was to assess the effects of a berry polyphenolic fraction (Orophenol®) composed of extracts from cranberry, wild blueberry, and strawberry on the main pathogenic determinants of P. gingivalis. Orophenol® attenuated the growth of P. gingivalis and decreased its hemolytic activity, its adherence to a basement membrane matrix model, and its proteinase activities. The berry polyphenolic fraction also impaired the production of reactive oxygen species (ROS) by oral keratinocytes stimulated with P. gingivalis. Lastly, using an in vitro model of oral keratinocyte barrier, the fraction exerted a protective effect against the damages mediated by P. gingivalis. In conclusion, the berry polyphenolic fraction investigated in the present study attenuated several pathogenic properties of P. gingivalis. Although future clinical investigations are required, our study provided evidence that the polyphenols contained in this fraction may represent bioactive molecules of high interest for the prevention and/or treatment of periodontal disease.

Alzheimer disease is one of the most frequent neurodegenerative diseases. Porphyromonas gingivalis is the key pathogen of chronic periodontitis, and it has a virulence factor known as gingipain. Gingipain is a proteolytic enzyme capable of penetrating the blood–brain barrier to reach the brain’s center of cognition. Gingipain that reaches the brain is suspected of having a relationship with an amyloid-beta plaque and neurofibrillary tangle, which play a critical role in the formation of Alzheimer disease. These bacteria may also suppress the immune system by reducing cytokine tumor growth factor (TGF-beta) and Interferon (IFN-gamma) and imbalance of Th17/Treg (Regulatory T cells). Besides that, the involvement of P. gingivalis in the brain may trigger neuroinflammation and lead to neuron defect and worsen Alzheimer disease. This review aims to discuss the correlation between P. gingivalis and the development of Alzheimer disease.

Abstract 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.

Introduction: Periodontitis is a very common polymicrobial infection of the oral cavity with wide systemic implications. It is influenced by multiple aspects, such as virulence of bacteria, the host response and resistance of bacteria to antibiotics, both within and outside the biofilm. Commonly, antibiotics are employed to break this vicious activity of microbes. There is a lacuna in the literature regarding the comparative efficacy of newer congeners of doxycycline. The aim of the study was to objectively compare the binding capacity of newer congeners of doxycycline with clinically significant targets relevant to periodontitis. Material and methods: A total of 5 drugs, viz. doxycycline, tigecycline, eravacycline, sarecycline and omadacycline, were selected, and molecular docking studies were performed with four targets: gingipain, FimA, interleukin-1ß and estrogen receptor ß. The studies were performed using AutoDock version 4. The results were reported based on the binding free energy, electrostatic interaction and intermolecular attraction. These values were compared and reported. Results: The drugs selected showed good binding to all four targets but had many differences in binding efficacy. Omadacycline, tigecycline, sarecycline, and doxycycline revealed 100% binding efficacy by occupying the core amino acid residues (444 HIS, 477 CYS and 388 ASP) over the target protein. Conclusions: Doxycycline can be replaced with omadacycline for clinical use. This result warrants future clinical investigations on omadacycline for periodontal therapy in both local and systemic administration. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, a Gram-negative anaerobic bacillus present in periodontal disease, is considered one of the major pathogens in periodontitis. A literature search for English original studies, case series and review articles published up to December 2019 was performed using the MEDLINE, PubMed and GoogleScholar databases, with the search terms "Porphyromonas gingivalis" AND the potentially associated condition or systemic disease Abstracts and full text articles were used to make a review of published research literature on P. gingivalis outside the oral cavity. The main points of interest of this narrative review were: (i) a potential direct action of the bacterium and not the systemic effects of the inflammatory acute-phase response induced by the periodontitis, (ii) the presence of the bacterium (viable or not) in the organ, or (iii) the presence of its virulence factors. Virulence factors (gingipains, capsule, fimbriae, hemagglutinins, lipopolysaccharide, hemolysin, iron uptake transporters, toxic outer membrane blebs/vesicles, and DNA) associated with P. gingivalis can deregulate certain functions in humans, particularly host immune systems, and cause various local and systemic pathologies. The most recent studies linking P. gingivalis to systemic diseases were discussed, remembering particularly the molecular mechanisms involved in different infections, including cerebral, cardiovascular, pulmonary, bone, digestive and peri-natal infections. Recent involvement of P. gingivalis in neurological diseases has been demonstrated. P. gingivalis modulates cellular homeostasis and increases markers of inflammation. It is also a factor in the oxidative stress involved in beta-amyloid production. [ABSTRACT FROM AUTHOR]

Alzheimer disease is one of the most frequent neurodegenerative diseases. Porphyromonas gingivalis is the key pathogen of chronic periodontitis, and it has a virulence factor known as gingipain. Gingipain is a proteolytic enzyme capable of penetrating the blood-brain barrier to reach the brain's center of cognition. Gingipain that reaches the brain is suspected of having a relationship with an amyloid-beta plaque and neurofibrillary tangle, which play a critical role in the formation of Alzheimer disease. These bacteria may also suppress the immune system by reducing cytokine tumor growth factor (TGF-beta) and Interferon (IFN-gamma) and imbalance of Th17/Treg (Regulatory T cells). Besides that, the involvement of P. gingivalis in the brain may trigger neuroinflammation and lead to neuron defect and worsen Alzheimer disease. This review aims to discuss the correlation between P. gingivalis and the development of Alzheimer disease. [ABSTRACT FROM AUTHOR]

Ingestion of Porphyromonas gingivalis , a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis , affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases. [Display omitted] • OMVs from Porphyromonas gingivalis increased permeability of Caco-2 monolayer. • Gingipains on OMVs is responsible for destruction of Caco-2 cell barrier. • Gingipains on OMVs degraded tight junction protein, occludin. • Porphyromonas gingivalis OMVs deliver gingipains into cytosol in Caco-2 cells. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet‐activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR‐specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp‐dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this. [ABSTRACT FROM AUTHOR]

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface‐expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg‐induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild‐type (W83) but not gingipain‐deficient (ΔK/R‐ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM‐1 and VE‐cadherin compared to infection with ΔK/R‐ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM‐1 or VE‐cadherin were systemically infected with W83 or ΔK/R‐ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain‐dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM‐1 and VE‐cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease. [ABSTRACT FROM AUTHOR]

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

The potential of probiotics on the prevention and control of periodontitis and other chronic inflammatory conditions has been suggested. Lactobacillus and Bifidobacterium species influence P. gingivalis interaction with gingival epithelial cells (GECs) but may not act in a unique way. In order to select the most appropriate probiotic against P. gingivalis, we aimed to evaluate the effect of several strains on Porphyromonas gingivalis biofilm formation and transcription virulence-associated factors (PgVAFs). Cell-free pH neutralized supernatants (CFS) and living Lactobacillus spp. and Bifidobacterium spp. were tested against P. gingivalis ATCC 33277 and W83, in mono- and multi-species (with Streptococcus oralis and S. gordonii) biofilms. Relative transcription of P. gingivalis genes (fimA, mfa1, kgp, rgp, ftsH and luxS) was determined in biofilms and under GECs co-infection. Probiotics CFS reduced P. gingivalis ATCC 33277 levels in mono-species biofilms and living probiotics reduced P. gingivalis abundance in multi-species biofilms. L. acidophilus LA5 down-regulated transcription of most PgVAFs in biofilms and GECs. Probiotics affect P. gingivalis biofilm formation by down-regulating overall PgVAFs with the most pronounced effect observed for L. acidophilus LA5. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The periodontal pathogen Porphyromonas gingivalis produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in the organism's virulence, and pigmentation. We previously showed that deletion of the PGN_0297 and PGN_0300 genes reduced the proteolytic activity of gingipains. The role of the PGN_0296 gene, consisting of an operon with the PGN_0297 and PGN_0300 genes, is unclear. Herein, we examined the effect of PGN_0296 gene deletion on the proteolytic activity. Although the proteolytic activity of the gingipains did not decrease in the culture supernatant of a PGN_0296 gene deletion mutant (ΔPGN_0296), the growth was delayed. [ABSTRACT FROM AUTHOR]

Epidemiological studies have identified an association between periodontitis and Alzheimer disease (AD); however, the nature of this association has been unclear. Recent work suggests that brain colonization by the periodontal pathogen Porphyromonas gingivalis may link these two inflammatory and degenerative conditions. Evidence of P. gingivalis infiltration has been detected in autopsy specimens from the brains of people with AD and in cerebrospinal fluid of individuals diagnosed with AD. Gingipains, a class of P. gingivalis proteases, are found in association with neurons, tau tangles, and beta-amyloid in specimens from the brains of individuals with AD. The brains of mice orally infected with P. gingivalis show evidence of P. gingivalis infiltration, along with various neuropathological hallmarks of AD. Oral administration of gingipain inhibitors to mice with established brain infections decreases the abundance of P. gingivalis DNA in brain and mitigates the neurotoxic effects of P. gingivalis infection. Thus, gingipain inhibition could provide a potential approach to the treatment of both periodontitis and AD. [ABSTRACT FROM AUTHOR]

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis -specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis. [ABSTRACT FROM AUTHOR]

Aim: To profile gingival tissue levels of human beta‐defensin (hBD)‐2 and hBD‐3 in relation to gingival inflammation, Th17‐related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities. Materials and Methods: Gingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real‐time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal–Wallis and Mann–Whitney U tests and Spearman correlation coefficients. Results: Subgingival plaque counts of P. gingivalis (p =.001) and gingipain activity (p <.001), as well as interleukin (IL)‐1β (p =.012), IL‐10 (p =.024), IL‐17A (p =.002), IL‐17F (p =.006), and IL‐23 (p =.036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD‐2 and hBD‐3 levels. Negative correlations were found between protease activity and hBD‐2 (p =.033) and hBD‐3(p =.003) levels. Conclusions: Shift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation. [ABSTRACT FROM AUTHOR]

Background and Objective The potential of probiotics on the prevention and control of periodontitis and other chronic inflammatory conditions has been suggested. Lactobacillus and Bifidobacterium species influence P. gingivalis interaction with gingival epithelial cells (GECs) but may not act in a unique way. In order to select the most appropriate probiotic against P. gingivalis, we aimed to evaluate the effect of several strains on Porphyromonas gingivalis biofilm formation and transcription virulence-associated factors (PgVAFs). Methods Cell-free pH neutralized supernatants (CFS) and living Lactobacillus spp. and Bifidobacterium spp. were tested against P. gingivalis ATCC 33277 and W83, in mono- and multi-species (with Streptococcus oralis and S. gordonii) biofilms. Relative transcription of P. gingivalis genes (fimA, mfa1, kgp, rgp, ftsH and luxS) was determined in biofilms and under GECs co-infection. Results Probiotics CFS reduced P. gingivalis ATCC 33277 levels in mono-species biofilms and living probiotics reduced P. gingivalis abundance in multi-species biofilms. L. acidophilus LA5 down-regulated transcription of most PgVAFs in biofilms and GECs. Conclusions Probiotics affect P. gingivalis biofilm formation by down-regulating overall PgVAFs with the most pronounced effect observed for L. acidophilus LA5.

Background and objectives 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. Results 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. Conclusions 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.

Porphyromonas gingivalis strains exhibit different phenotypes in vitro , different virulence potential in animal models, and different associations with human diseases, with strains classified as virulent/more virulent (e.g., A7436 and W83) or as less virulent/avirulent (e.g., ATCC 33277). In this study, we comparatively analyzed the A7436 and ATCC 33277 strains to better understand their variability. Global gene expression analysis in response to heme and iron limitation revealed more pronounced differences in the A7436 than in the ATCC 33277 strain; however, in both strains, the largest changes were observed in genes encoding hypothetical proteins, genes whose products participate in energy metabolism, and in genes encoding proteins engaged in transport and binding proteins. Our results confirmed that variability between P. gingivalis strains is due to differences in the arrangement of their genomes. Analysis of gene expression of heme acquisition systems demonstrated that not only the availability of iron and heme in the external environment but also the ability to store iron intracellularly can influence the P. gingivalis phenotype. Therefore, we assume that differences in virulence potential may also be due to differences in the production of systems involved in iron and heme acquisition, mainly the Hmu system. In addition, our study showed that hemoglobin, in a concentration-dependent manner, differentially influences the virulence potential of P. gingivalis strains. We conclude that iron and heme homeostasis may add to the variability observed between P. gingivalis strains., Importance: Periodontitis belongs to a group of multifactorial diseases, characterized by inflammation and destruction of tooth-supporting tissues. P. gingivalis is one of the most important microbial factors involved in the initiation and progression of periodontitis. To survive in the host, the bacterium must acquire heme as a source of iron and protoporphyrin IX. P. gingivalis strains respond differently to changing iron and heme concentrations, which may be due to differences in the expression of systems involved in iron and heme acquisition. The ability to accumulate iron intracellularly, being different in more and less virulent P. gingivalis strains, may influence their phenotypes, production of virulence factors (including proteins engaged in heme acquisition), and virulence potential of this bacterium., Competing Interests: The authors declare no conflict of interest.

Porphyromonas gingivalis, a keystone pathogen in periodontitis, secretes an array of virulence factors including gingipains via the type IX secretion system (T9SS). Inactivation of any component of the T9SS leads to the accumulation of secreted proteins in unprocessed and, in the case of progingipains, inactive forms in the periplasm. To cast light on the paradox that active gingipains are essential for P. gingivalis fitness in vivo but a functional T9SS is not (Frontiers in Cellular and Infection Microbiology, 2017, 7:378), we have compared virulence of wild-type P. gingivalis W83 and the gingipain-null strain with isogenic mutants deficient in individual T9SS components. Using an in vivo subcutaneous chamber mouse model of infection, gingipain-null strain secretion mutants showed no virulence, but their pathogenic potential was reconstituted by coinfection with a low number of the parental strain. Apparently the same mechanism compensated fitness of mutants lacking functional T9SS the transposon library. In contrast to the parental strain, all mutants elicited significantly lower but an effective inflammatory immune response, which cleared infection and prevented systemic dissemination of P. gingivalis to organs. There were no significant differences in immune responses to different secretion mutants, which were generally more stimulatory than the gingipain-null strain. Together, these results indicate that functional T9SS is essential for P. gingivalis virulence apparently through delivery of active gingipains to the bacterial surface. Therefore, T9SS is a legitimate target for drug development to treat periodontitis. [ABSTRACT FROM AUTHOR]

Abstract Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus , with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra , S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 h caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signalling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm. Highlights • Parvimonas micra enhance growth of Porphyromonas gingivalis in 10% serum. • P. micra significantly enhance gingipain activity in multispecies consortia. • P. micra secretes glycolytic enzymes into the external environment. • Gingipains are upregulated by soluble factors released from P. micra. [ABSTRACT FROM AUTHOR]

Introduction: Periodontal diseases (PD) are complex oral inflammatory diseases initiated by keystone bacteria such as Porphyromonas gingivalis. A vaccine for PD is desirable as clinical treatment involves protracted maintenance strategies aimed to retain dentition. Although prior immunization approaches targeting P. gingivalis have reported variable success in limiting facets of disease such as oral bone loss, it remains that a vaccine for this disease may be attainable. Aim: To investigate cell‐free protein synthesis (CFPS) as a platform to produce vaccinable targets suitable for efficacy testing in a P. gingivalis‐induced murine oral bone loss model. Materials and Methods: Recombinantly generated P. gingivalis minor fimbriae protein (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were combined in equivalent doses in adjuvants and injected intramuscularly to immunize mice. Serum levels of protein‐specific antibody were measured by ELISA, and oral bone levels were defined by morphometrics. Results: Recombinantly generated P. gingivalis proteins possessed high fidelity to predicted size and elicited protein‐specific IgG following immunization. Importantly, immunization with the vaccine cocktail protected from P. gingivalis elicited oral bone loss. Conclusion: These data verify the utility of the CFPS technology to synthesize proteins that have the capacity to serve as novel vaccines. [ABSTRACT FROM AUTHOR]

Interaction between two periodontal pathogens, Porphyromonas gingivalis and Treponema denticola, contributes to plaque biofilm formation. Porphyromonas gingivalis forms aggregates with T. denticola through its adhesion/hemagglutinin domain (Hgp44). In this study, we investigated the specific domain of P. gingivalis Hgp44 responsible for adhesion to T. denticola using expression vectors harboring P. gingivalis Hgp44 DNA sequences encoding amino acid residues 1–419. Six plasmids harboring fragments in this region were generated by PCR amplification and self-ligation, and recombinant proteins r-Hgp44 (residues 1–419), r-Hgp441 (residues 1–124), r-Hgp442 (1–199), r-Hgp443 (1–316), r-Hgp444 (199–419), r-Hgp445 (124–198) and r-Hgp446 (199–316) were produced, as confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting. r-Hgp44, r-Hgp443 and r-Hgp446 showed greater adhesion to T. denticola sonicates than the control, as determined by enzyme-linked immunosorbent assay. r-Hgp446 reduced the coaggregation of P. gingivalis and T. denticola. Scanning electron and confocal laser scanning microscopy analyses revealed that r-Hgp446 reduced dual-species biofilm formation. Our results indicate that residues 199–316 of P. gingivalis Hgp44 are mainly responsible for adhesion to T. denticola; inhibiting this domain could potentially disrupt periodontopathic biofilm formation and maturation. Porphyromonas gingivalis Hgp44 residues 199–316 are responsible for adhesion to Treponema denticola; inhibition of this domain may disrupt periodontopathic biofilm formation and maturation. [ABSTRACT FROM AUTHOR]