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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 (Pearsons 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 Alzheimers disease.

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.

Syfte: Syftet med denna studie var att experimentellt bestämma om Parvimonas micra ökar tillväxt och gingipainaktiviteten hos olika stammar av Porphyromonas gingivalis. En ökning av gingipainaktiviteten indikerar att P. micra skulle kunna öka virulensen i P. gingivalis.Material och metod: Fem stammar av P. gingivalis 1A, 16A, 50A, 33F och W50 odlades med P. micra och jämfördes med kontrollen som endast innehöll P. gingivalis. Arginin gingipain (Rgp) och lysin gingipain (Kgp) aktivitet mättes med hjälp av Bikkam-16 respektive Bikkam-14. Bakterietillväxt mättes genom att mäta optisk densitet.Resultat: Experimenten visade en ökad tillväxt av stammarna 1A, 16A och 50A när de odlades med P. micra, 33F visade måttlig tillväxt och W50 ingen tillväxt.Rgp aktiviteten ökade i vissa P. gingivalis-stammar (1A och 16A) i närvaro P. micra men ökade inte i andra (W50, 33F och 50A)Kgp aktiviteten ökade i P. gingivalis lA i närvaro av P. micra men ingen ökning skedde i de andra stammarna (16A, W50, 33F och 50A).Slutsats: Resultaten från denna studie visade att P. micra påverkar tillväxten och gingipainaktiviteten hos vissa stammar av P. gingivalis men inte alla., Aim: The aim of this study was to experimentally determine if Parvimonas micra increase growth and gingipain activity in different strains of Porphyromonas gingivalis. An increase in gingipain activity could indicate that P. micra induces pathogenic expression of P. gingivalis.Material and method: Five strains of P. gingivalis 1A, 16A, 50A, 33F and W50 was cultivated together with P. micra while control samples contained only P. gingivalis. Activity of arginine gingipain (Rgp) and lysine gingipain (Kgp) was measured using Bikkam-16 respectively Bikkam-14. Bacterial growth was determining by measuring the optical density.Results: The experiments show an increased growth of some strains (1A, 16A and 50A) of P. gingivalis when grown with P. micra, one showed moderate growth (33F) and one showed no growth (W50).The gingipain activity (Rgp) increased in P. gingivalis strains (1A and 16A) in the presence of P. micra but did not increase in other strains (W50, 33F and 50A)The gingipain activity (Kgp) increased in one P. gingivalis strains (1A) when mixed with P. micra and did not increase in others (16A, W50, 33F and 50A). Conclusion: The results from this study indicates that P. micra effect the growth and gingipain activity in certain strains of P. gingivalis but not all.

Syfte: Syftet med denna studie var att experimentellt bestämma om Parvimonas micra ökar tillväxt och gingipainaktiviteten hos olika stammar av Porphyromonas gingivalis. En ökning av gingipainaktiviteten indikerar att P. micra skulle kunna öka virulensen i P. gingivalis.Material och metod: Fem stammar av P. gingivalis 1A, 16A, 50A, 33F och W50 odlades med P. micra och jämfördes med kontrollen som endast innehöll P. gingivalis. Arginin gingipain (Rgp) och lysin gingipain (Kgp) aktivitet mättes med hjälp av Bikkam-16 respektive Bikkam-14. Bakterietillväxt mättes genom att mäta optisk densitet.Resultat: Experimenten visade en ökad tillväxt av stammarna 1A, 16A och 50A när de odlades med P. micra, 33F visade måttlig tillväxt och W50 ingen tillväxt.Rgp aktiviteten ökade i vissa P. gingivalis-stammar (1A och 16A) i närvaro P. micra men ökade inte i andra (W50, 33F och 50A)Kgp aktiviteten ökade i P. gingivalis lA i närvaro av P. micra men ingen ökning skedde i de andra stammarna (16A, W50, 33F och 50A).Slutsats: Resultaten från denna studie visade att P. micra påverkar tillväxten och gingipainaktiviteten hos vissa stammar av P. gingivalis men inte alla., Aim: The aim of this study was to experimentally determine if Parvimonas micra increase growth and gingipain activity in different strains of Porphyromonas gingivalis. An increase in gingipain activity could indicate that P. micra induces pathogenic expression of P. gingivalis.Material and method: Five strains of P. gingivalis 1A, 16A, 50A, 33F and W50 was cultivated together with P. micra while control samples contained only P. gingivalis. Activity of arginine gingipain (Rgp) and lysine gingipain (Kgp) was measured using Bikkam-16 respectively Bikkam-14. Bacterial growth was determining by measuring the optical density.Results: The experiments show an increased growth of some strains (1A, 16A and 50A) of P. gingivalis when grown with P. micra, one showed moderate growth (33F) and one showed no growth (W50).The gingipain activity (Rgp) increased in P. gingivalis strains (1A and 16A) in the presence of P. micra but did not increase in other strains (W50, 33F and 50A)The gingipain activity (Kgp) increased in one P. gingivalis strains (1A) when mixed with P. micra and did not increase in others (16A, W50, 33F and 50A). Conclusion: The results from this study indicates that P. micra effect the growth and gingipain activity in certain strains of P. gingivalis but not all.

Periodontal pathogens, including Porphyromonas gingivalis, can form biofilms in dental pockets and cause inflammation, which is one of the underlying mechanisms involved in the development of periodontal disease, ultimately leading to tooth loss. Although P. gingivalis is protected in the biofilm, it can still cause damage and modulate inflammatory responses from the host, through secretion of microvesicles containing proteinases. The aim of this study was to evaluate the role of cysteine proteinases in P. gingivalis colony growth and development, and subsequent immunomodulatory effects on human gingival fibroblast. By comparing the wild type W50 with its gingipain deficient strains we show that cysteine proteinases are required by P. gingivalis to form morphologically normal colonies. The lysine-specific proteinase (Kgp), but not arginine-specific proteinases (Rgps), was associated with immunomodulation. P. gingivalis with Kgp affected the viability of gingival fibroblasts and modulated host inflammatory responses, including induction of TGF-β1 and suppression of CXCL8 and IL-6 accumulation. These results suggest that secreted products from P. gingivalis, including proteinases, are able to cause damage and significantly modulate the levels of inflammatory mediators, independent of a physical host-bacterial interaction. This study provides new insight of the pathogenesis of P. gingivalis and suggests gingipains as targets for diagnosis and treatment of periodontitis.

The interaction between human gingival fibroblasts (HGFs) and Porphyromonas gingivalis plays an important role in the development and progression of periodontitis. Porphyromonas gingivalis possesses several virulence factors, including cysteine proteases, the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains. Studying the mechanisms that P.gingivalis, and its derived virulence, use to propagate and interact with host cells will increase the understanding of the development and progression of periodontitis. In this study, we aimed to elucidate how P.gingivalis influences the inflammatory events in HGFs regarding transforming growth factor-(1) (TGF-(1)), CXCL8, secretory leucocyte protease inhibitor (SLPI), c-Jun and indoleamine 2,3-dioxygenase (IDO). HGFs were inoculated for 6 and 24h with the wild-type strains ATCC 33277 and W50, two gingipain-mutants of W50 and heat-killed ATCC 33277. The P.gingivalis regulated CXCL8 and TGF-(1) in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P<0.001) and low levels of TGF-(1). We show that HGFs express and secrete SLPI, which was significantly suppressed by P.gingivalis (P<0.05). This suggests that by antagonizing SLPI, P.gingivalis contributes to the tissue destruction associated with periodontitis. Furthermore, we found that P.gingivalis inhibits the expression of the antimicrobial IDO, as well as upregulating c-Jun (P<0.05). In conclusion, P.gingivalis both triggers and suppresses the immune response in HGFs. Consequently, we suggest that the pathogenic effects of P.gingivalis, and especially the activity of the gingipains on the inflammatory and immune response of HGFs, are crucial in periodontitis., Funding Agencies:Foundation of Olle EngkvistKnowledge Foundation

Periodontal pathogens, including Polphyromonas gingivalis, can form biofilms in dental pockets and cause inflammation, which is one of the underlying mechanisms involved in the development of periodontal disease, ultimately leading to tooth loss. Although P. gingivalis is protected in the biofilm, it can still cause damage and modulate inflammatory responses from the host, through secretion of microvesicles containing proteinases. The aim of this study was to evaluate the role of cysteine proteinases in P. gingivalis colony growth and development, and subsequent immunomodulatory effects on human gingival fibroblast. By comparing the wild type W50 with its gingipain deficient strains we show that cysteine proteinases are required by P. gingivalis to form morphologically normal colonies. The lysine-specific proteinase (Kgp), but not arginine-specific proteinases (Rgps), was associated with immunomodulation. P. gingivalis with Kgp affected the viability of gingival fibroblasts and modulated host inflammatory responses, including induction of TGF-beta 1 and suppression of CXCL8 and IL-6 accumulation. These results suggest that secreted products from P. gingivalis, including proteinases, are able to cause damage and significantly modulate the levels of inflammatory mediators, independent of a physical host-bacterial interaction. This study provides new insight of the pathogenesis of P. gingivalis and suggests gingipains as targets for diagnosis and treatment of periodontitis., Funding Agencies:Swedish Heart and Lung AssociationFoundation of Olle Engkvist

Porphyromonas gingivalis is an important etiological agent of periodontal disease - a disease that affects an estimated 49,000,000 people in the United States of America. Periodontal disease includes gingivitis – inflammation of the gums, and periodontitis – destruction of the teeth and their supporting tissues. Porphyromonas gingivalis is associated with the chronic form of periodontal disease in addition to several systemic diseases. The vimA gene of P. gingivalis has been previously shown to play a significant role in the biogenesis of gingipains (trypsin-like cysteine proteases). The vimA has also been demonstrated to play a role in hemolysis, hemagglutination, autoaggregation, posttranslational protein modification, and LPS biogenesis. Though significant progress has been made in characterizing this gene, much remained unsolved prior to this work, especially as it relates to the molecular mechanisms and interactions of this protein in surface biogenesis, transport and membrane integrity. The focus of our work was to clarify the role of VimA on cell surface biogenesis. We were able to show via TEM and AFM that there were significant differences between the vimA-defective mutant (FLL92) and the wild-type (W83). Particularly, the topography of FLL92 showed numerous fine structures compared with W83, and had an irregular, loosely bound capsule. Fimbrial synthesis was also affected by the VimA mutation. FimA coupled immunogold particles localized with the abundant fimbrial appendages in FLL92, compared with only a few adhering in W83. Lectins were used to clarify the effect of the vimA mutation on the glycosylation of outer-membrane proteins. Outer membrane proteins glycosylated with Galactose (β 1,3) N-Acetylgalactosamine, N-acetyl-α-D-galactosamine, Galactose (β 1,4) N-Acetylglucosamine, N-acetyl-D-galactosamine and Sialic Acid (N-Acetyl neuramic acid) were affected by the vimA mutation. Mass Spectrometric analysis of outermembrane and extracellular proteins ident

Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum., PNAS, 107(1), pp.276-281; 2010

Introduction: Human cells express membrane-bound complement regulatory proteins to prevent complement-mediated autologous tissue damage. In this study, we hypothesized that Porphyromonas gingivalis, the major etiological agent of chronic periodontitis, causes the shedding or proteolysis of the complement regulatory protein CD46 expressed by oral epithelial cells. Methods: Oral epithelial cells were treated with a culture of P. gingivalis before measurement of membrane-bound and shed CD46 by enzyme-linked immunosorbent assay (ELISA). The effect of soluble recombinant CD46 on secretion of interleukin-8 (IL-8) by epithelial cells was evaluated by ELISA. The susceptibility of soluble recombinant CD46 to proteolytic degradation by cells and purified Lys-gingipain of P. gingivalis was investigated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/western immunoblotting analysis. Results: Oral epithelial cells treated with a culture of P. gingivalis showed a lower reactivity with antibodies directed to CD46. ELISA revealed that such a treatment resulted in increased amounts of CD46 in the conditioned media suggesting that P. gingivalis caused the shedding of membrane-anchored CD46. Stimulation of epithelial cells with soluble recombinant CD46 induced IL-8 secretion in a dose-dependent manner. Whole cells and purified Lys-gingipain of P. gingivalis degraded recombinant CD46 in a dose-dependent manner. Conclusion: This study showed the ability of P. gingivalis to induce the shedding/ proteolysis of CD46 from the surface of oral epithelial cells. This may render host cells susceptible to the complement system and contribute to tissue damage and the inflammatory process in periodontitis

Objective: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. Material and Methods: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (CT) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. Results: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (CT) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. Conclusion: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples

Objective: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. Material and Methods: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (CT) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. Results: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (CT) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. Conclusion: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples