The role of phagocytosis,oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram‐negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild‐type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild‐type strain ATCC33277, whereas the other wild‐type strain W50 was less effective. Quantitative real‐time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.     

Lipoprotein modifications by gingipains of Porphyromonas gingivalis

Background and Objective

Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low‐density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.

Material and Methods

Plasma lipoproteins were isolated from whole blood treated with wild‐type and gingipain‐mutant (lacking either the Rgp‐ or Kgp gingipains) P. gingivalis by density/gradient‐ultracentrifugation and were studied using 2‐dimensional gel electrophoresis followed by matrix‐assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis‐induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid‐reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.

Results

Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB‐100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high‐density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.

Conclusion

Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.     

Effect of different Aggregatibacter actinomycetemcomitans strains on dual-species biofilms formed with Porphyromonas gingivalis or Dialister pneumosintes

There are five evolutionarily divergent clades of Aggregatibacter actinomycetemcomitans, with possible differences in phenotype and virulence potential among strains. This study examined the formation of biofilm by each of 11 distinct strains of A. actinomycetemcomitans, alone or after coculture with two species of oral bacteria (Porphyromonas gingivalis ATCC33277 or Dialister pneumosintes ATCC33048). Confocal laser scanning microscopy (CLSM) and electron microscopy were used to characterize the dual-species biofilms of interest. A reduction in dual-species A. actinomycetemcomitans–P. gingivalis biofilms was observed for A. actinomycetemcomitans RHAA1, suggesting an antagonistic relationship. The amounts of dual-species A. actinomycetemcomitans–D. pneumosintes biofilms were either increased or decreased in some – but not all – strains, indicative of strain-specific phenotypes. The CLSM analyses confirmed the existence of an antagonistic relationship between A. actinomycetemcomitans D7S-1 and P. gingivalis ATCC33277, and a synergistic relationship between A. actinomycetemcomitans D7S-1 and D. pneumosintes ATCC33048. The electron microscopy analyses revealed distinct morphological features of A. actinomycetemcomitans D7S-1 and D. pneumosintes ATCC33048 dual-species biofilms. The results indicate that the relationship between A. actinomycetemcomitans and oral bacteria may vary among strains, which could lead to distinct strain-specific patterns of niche sharing in subgingival microbiota.     

Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis

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‐β₁ (TGF‐β₁), CXCL8, secretory leucocyte protease inhibitor (SLPI), c‐Jun and indoleamine 2,3‐dioxygenase (IDO). HGFs were inoculated for 6 and 24 h 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‐β₁ in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P < 0.001) and low levels of TGF‐β₁. 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.     

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll‐like receptors (TLR) and protease‐activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet–bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg‐gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1‐agonist Pam₃CSK₄, which evoked a time‐dependent release of the chemokine. Interestingly, the TLR2/1‐evoked response was abolished by a following addition of viable P. gingivalis wild‐types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam₃CSK₄‐stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor‐1, and that also these responses were antagonized by P. gingivalis. These results supports immune‐modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.     

Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis

We have previously shown that benzamidine‐type compounds can inhibit the activity of arginine‐specific cysteine proteinases (gingipains HRgpA and RgpB); well‐known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important periodontopathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, because pentamidine, which is a 20‐fold less efficient inhibitor of gingipain than 2,6‐bis‐(4‐amidinobenzyl)‐cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine‐derived compounds other than Arg‐gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5‐1‐2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine–Sepharose column to identify proteins interacting with benzamidine. In addition to HRgpA and RgpB the analysis revealed heat‐shock protein GroEL as another ligand for benzamidine. To better understand the effect of benzamidine‐derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine‐derived compounds caused significant increases in GroEL, at both the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also by pentamidine, a relatively weak gingipain inhibitor. This effect may depend not only on gingipain inhibition but also on interaction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment.     

Gingipain‐dependent augmentation by Porphyromonas gingivalis of phagocytosis of Tannerella forsythia

In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that manipulates host immune responses leading to dysbiotic oral microbial communities. Arg‐gingipains (RgpA and RgpB) and Lys‐gingipain (Kgp) are responsible for the majority of bacterial proteolytic activity and play essential roles in bacterial virulence. Therefore, gingipains are often considered as therapeutic targets. This study investigated the role of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in a multiplicity of infection‐dependent and gingipain‐dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in mixed infection, and this coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, the direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected macrophages, more T. forsythia remained in cells coinfected with gingipain‐expressing P. gingivalis than in cells coinfected with the gingipain‐null mutant or infected only with T. forsythia at 24 and 48 h post‐infection. Collectively, these results suggest that P. gingivalis, mainly via its gingipains, alters the clearance of T. forsythia, and provide some insights into the role of P. gingivalis as a keystone pathogen.     

Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co‐aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.     

Identification of gingipain‐specific I‐Ab‐restricted CD4+ T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice

Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4⁺ T‐cell‐mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I‐A^b‐binding peptides existing in Kgp and RgpA are presented to CD4⁺ T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I‐A^b‐binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide‐binding groove of the I‐A^b molecule of C57BL/6 mice. An ELISpot‐based screen revealed those peptide‐primed effector/memory CD4⁺ T cells that could be re‐stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin‐17A or interferon‐γ. Two immunodominant peptides, Kgp_467–477 (pKgp) and RgpA_1054–1064/Kgp_1074–1084 (pR/Kgp) were identified and engineered to be displayed on I‐A^b molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin‐conjugated pKgp::I‐A^b and pR/Kgp::I‐A^b tetramers. We found that only pR/Kgp::I‐A^b bound with the desired specificity to gingipain‐specific CD4⁺ T cells. The pR/Kgp::I‐A^b tetramer complex will allow the identification of effector/memory CD4⁺ T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.     

Heme acquisition mechanisms of Porphyromonas gingivalis – strategies used in a polymicrobial community in a heme‐limited host environment

Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell‐surface heme‐containing pigment in the form of μ‐oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB‐dependent outer‐membrane receptors (HmuR, HusB, IhtA), and hemophore‐like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)‐containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine‐ and lysine‐specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.     

Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line

Guentsch A, Rönnebeck M, Puklo M, Preshaw PM, Pfister W, Eick S. Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01224.x. © 2009 John Wiley & Sons A/S Background and Objective: The purpose of this study was to investigate the influence of serum on the interaction of periodontal pathogens with epithelial cells using an epithelial cell line (KB cells). This is important because serum is a key component of gingival crevicular fluid and may influence inflammatory responses in epithelial cells exposed to periodontal pathogens. Material and Methods: Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 were co‐cultured with KB cells either with or without the addition of up to 10% human serum or 50 mg/mL human serum albumin. The numbers of free‐floating, adherent and intracellular bacteria were determined up to 18 h after exposure of the epithelial cells to the pathogens. Additionally, the concentrations of interleukin (IL)‐6 and IL‐8 produced by the epithelial cells in response to exposure to the bacteria were determined. Results: Serum and human serum albumin reduced the number of internalized A. actinomycetemcomitans Y4 organisms in the epithelial cells, increased the levels of IL‐6 and IL‐8 in the supernatants of infected cells (those with internalized A. actinomycetemcomitans) and influenced non‐infected epithelial cells. Increased IL‐6 and IL‐8 concentrations were also detected in the supernatants of KB cells infected with P. gingivalis ATCC 33277. Interleukin‐6 and IL‐8 were detectable after addition of serum, probably as a result of inhibition of the activity of P. gingivalis cysteine proteinases by serum. Conclusion: Serum promotes the release of the cytokines IL‐6 and IL‐8 by epithelial cells. This mechanism is influenced by periodontal pathogens and may maintain clinical periodontal inflammation.     

In vivo expression of proteases and protease inhibitor,a serpin,by periodontal pathogens at teeth and implants

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK‐proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri‐implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri‐implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri‐implantitis sites (n = 10). Concentration of interleukin‐8 (IL‐8), IL‐1β and IL‐10 in GCF was determined by enzyme‐linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT‐PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL‐8 and IL‐1β, were associated with disease severity in the periodontal and peri‐implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK‐protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin‐1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK‐proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri‐implant diseases.     

Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation

Asahi Y, Noiri Y, Igarashi J, Asai H, Suga H, Ebisu S. Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01228.x © 2009 John Wiley & Sons A/S Background and Objective: The gram‐negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram‐negative bacteria is regulated by a quorum‐sensing circuit that relies on N‐acyl homoserine lactones (HSLs). Some synthetic N‐acyl HSL analogues act as quorum‐sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N‐acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. Material and Methods: A flow‐cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N‐acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. Results: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm‐forming cells (p < 0.05), and these biofilm structures were less well formed three‐dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue‐treated groups. Conclusion: Three synthetic N‐acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.     

Gingipain‐dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion

Porphyromonas gingivalis and Tannerella forsythia are gram‐negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4‐h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis‐mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p‐4E‐BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection.     

Porphyromonas gingivalis-induced Platelet Aggregation in Human Plasma

The periodontal pathogen Porphyromonas gingivalis has the ability to aggregate human platelets. In this study, the mechanism of P. gingivalis-induced platelet aggregation in platelet-rich plasma (PRP) was investigated. Proteinase inhibitors toward Arg- and Lys-specific gingipain (Rgp and Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP and it was found that proteolytic activity of gingipains did not contribute to P. gingivalis-induced platelet aggregation in plasma. The study using mutant strains revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and hemagglutinin A (HagA) -encoding genes that intragenically coded for adhesin Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP P. gingivalis cell-reactive IgG in plasma, and FcyRIIa receptor and to a lesser extent GP Ibα receptor on platelets were found to be essential for P. gingivalis-induced platelet aggregation in PRP These results reveal a novel mechanism of platelet aggregation by P. gingivalis and contribute to elucidation of the common nature of the interaction between platelets and pathogenic bacteria.     

Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S‐) layer modified with a unique O‐glycan. Both the S‐layer and the O‐glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S‐layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony‐forming unit counts and quantitative real‐time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S‐layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S‐layer in the positioning of this species within the biofilm, its co‐localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.     

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A‐LPS

Porphyromonas gingivalis is a Gram‐negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)‐protoporphyrin IX] or hemin [Fe(III)‐protoporphyrin IX‐Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin‐binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine‐proteases Arg‐ and Lys‐gingipains and two lipopolysaccharides (LPS), O‐LPS and A‐LPS. The gingipains are required for the production of the black pigment, μ‐oxo‐bisheme {[Fe(III)PPIX]₂ O}, which is derived from hemoglobin and deposited on the bacterial cell‐surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of μ‐oxo‐bisheme on the cell‐surface. A P. gingivalis mutant defective in the biosynthesis of Arg‐gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys‐gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α‐1, 3‐mannosyltransferase) produce non‐pigmented colonies. However, only those mutants lacking A‐LPS showed reduced hemin‐binding when cells in suspension were incubated with hemin. Using native, de‐O‐phosphorylated and de‐lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin‐binding to O‐polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin‐binding to A‐PS. We conclude that A‐LPS in the outer‐membrane of P. gingivalis serves as a scaffold/anchor for the retention of μ‐oxo‐bisheme on the cell surface and pigmentation is dependent on the presence of A‐LPS.     

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺‐promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺‐promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl₂ exhibited enhanced cell aggregation and increased levels of cell‐associated Ca²⁺. Biofilm‐derived cells grown in the presence of Ca²⁺ exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca²⁺‐promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca²⁺‐promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.