Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

The ability for DNA mismatch repair, after oxidative stress‐induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress‐induced DNA damage involving 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) may occur by a yet‐to‐be described mechanism in P. gingivalis. 8‐oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF‐ermAM antibiotic cassette and used to create a mutY‐deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild‐type W83 strain. DNA oligomers containing a site‐specific 8‐oxoG:A mispair was repaired similarly in both the P. gingivalis mutY‐defective mutant and wild‐type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8‐oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8‐oxoG:A mismatch in this organism.     

Scavenger receptor A is expressed by macrophages in response to Porphyromonas gingivalis,and participates in TNF‐α expression

Introduction: Porphyromonas gingivalis is a periodontopathic bacterium closely associated with generalized aggressive periodontal disease. Pattern recognition receptors (PRRs) participate in host response to this organism. It is likely that PRRs not previously recognized as part of the host response to P. gingivalis also participate in host response to this organism. Methods and Results: Employing qRT‐PCR, we observed increased msr1 gene expression at 2, 6, and 24 h of culture with P. gingivalis strain 381. Flow cytometry revealed increased surface expression of SR‐A protein by the 24 h time point. Macrophages cultured with an attachment impaired P. gingivalis fimA‐ mutant (DPG3) expressed intermediate levels of SR‐A expression. Heat‐killed P. gingivalis stimulated SR‐A expression similar to live bacteria, and purified P. gingivalis capsular polysaccharide stimulated macrophage SR‐A expression, indicating that live whole organisms are not necessary for SR‐A protein expression in macrophage response. As SR‐A is known to play a role in lipid uptake by macrophages, we tested the ability of low‐density lipoprotein (LDL) to influence the SR‐A response of macrophages to P. gingivalis, and observed no effect of LDL on P. gingivalis‐elicited SR‐A expression. Lastly, we observed that SR‐A knockout (SR‐A^?/?) mouse macrophages produced significantly more tumor necrosis factor (TNF)‐α than wild type mouse macrophages cultured with P. gingivalis. Conclusion: These data identify that SR‐A is expressed by macrophages in response to P. gingivalis, and support that this molecule plays a role in TNF‐α production by macrophages to this organism.     

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. A saliva-coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell-cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may, therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.     

Comparing culture, real-time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri-implant infections

Galassi F, Kaman WE, Anssari Moin D, van der Horst J, Wismeijer D, Crielaard W, Laine ML, Veerman ECI, Bikker FJ, Loos BG. Comparing culture, real‐time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri‐implant infections. J Periodont Res 2012; 47: 616–625. © 2012 John Wiley & Sons A/S Background and Objective: The aim of the study was to compare the detection of Porphyromonas gingivalis using a fluorescence resonance energy transfer (FRET) technology with commonly used diagnostic methods in salivary and subgingival plaque samples from subjects with dental implants. P. gingivalis was considered as a marker for a pathogenic microbiota. Material and Methods: Ninety‐seven adult subjects were recruited, including periodontally healthy controls with no dental implants, implant controls with no peri‐implant disease and patients with peri‐implant disease. Saliva and subgingival/submucosal plaque samples were collected from all subjects and were analyzed using culture, real‐time PCR and FRET technology employing P. gingivalis‐specific substrates. Results: It was found that the P. gingivalis‐specific substrates were highly suitable for detecting the presence of P. gingivalis in saliva and in subgingival plaque samples, showing comparable specificity to culture and real‐time PCR. Conclusion: We applied the FRET technology to detect P. gingivalis in implant patients with or without an implant condition and in controls without implants. The technique seems suitable for detection of P. gingivalis in both plaque and saliva samples. However, with all three techniques, P. gingivalis was not very specific for peri‐implantitis cases. Future work includes fine‐tuning the FRET technology and also includes the development of a chair‐side application.     

Effect of Streptococcus sanguinis/Porphyromonas gingivalis single and combined biofilms upon platelet aggregation

Oral Diseases (2012) 18, 586–594 Objective: To assess the effect of two oral bacteria Streptococcus sanguinis and Porphyromonas gingivalis upon platelet aggregation. Materials and Methods: Streptococcus sanguinis, P. gingivalis, S. sanguniis + P. gingivalis were added to platelet‐rich plasma and platelet aggregation measured using a platelet aggregometer. Platelets were passed through a flow chamber with S. sanguinis, P. gingivalis or a biofilm of S. sanguinis and P. gingivalis coated with saliva. Platelet adhesion to the chamber was observed under a fluorescence microscope for 15 min. The positive control was platelets treated with adrenaline; the negative control was platelets treated with phosphate‐buffered saline. Results: The mean (± s.e.) aggregation magnitude of S. sanguinis and P. gingivalis was 77.7 ± 7.4% and 79.3 ± 9.9%, respectively. The aggregation magnitude of S. sanguinis + P. gingivalis was 51.3 ± 12.9%, which was significantly lower than that for S. sanguinis/P. gingivalis (P < 0.05). In the flow chamber system, platelets adhered to S. sanguinis/P.gingivalis respectively within 3 min, and reached a plateau at 5–15 min. Under the condition of the S. sanguinis‐ and P. gingivalis‐saliva biofilm, platelet adhesion to the biofilm was significantly reduced at 5–15 min (P < 0.05). Conclusions: In the static or dynamic flow system, platelets adhered to S. sanguinis or P. gingivalis. However, if S. sanguinis was mixed with P. gingivalis, the aggregation magnitude (%) was significantly reduced.     

The possible mechanism of preterm birth associated with periodontopathic Porphyromonas gingivalis

Hasegawa‐Nakamura K, Tateishi F, Nakamura T, Nakajima Y, Kawamata K, Douchi T, Hatae M, Noguchi K. The possible mechanism of preterm birth associated with periodontopathic Porphyromonas gingivalis. J Periodont Res 2011; 46: 497–504. © 2011 John Wiley & Sons A/S Background and Objective: Previous studies have shown that Porphyromonas gingivalis is found in the amniotic fluid and placentae of pregnant women with some obstetric diseases. However, the biological effects of P. gingivalis on intrauterine tissues remain unclear. The aim of this study was to investigate the presence of P. gingivalis in chorionic tissues from hospitalized high‐risk pregnant women, and the effects of P. gingivalis lipopolysaccharide on the production of proinflammatory molecules in human chorion‐derived cells. Material and Methods: Twenty‐three subjects were selected from Japanese hospitalized high‐risk pregnant women. The presence of P. gingivalis in chorionic tissues was analyzed by PCR. Cultured chorion‐derived cells or Toll‐like receptor‐2 (TLR‐2) gene‐silenced chorion‐derived cells were stimulated with P. gingivalis lipopolysaccharide. Real‐time PCR was performed to evaluate TLR‐2 and Toll‐like receptor‐4 (TLR‐4) mRNA expression in the cells. Levels of interleukin‐6 and interleukin‐8 in culture supernatants of the chorion‐derived cells were measured by ELISA. Results: P. gingivalis DNA was detected in chorionic tissues from two women with threatened preterm labor, two with multiple pregnancy and two with placenta previa. Stimulation of chorion‐derived cells with P. gingivalis lipopolysaccharide significantly increased TLR‐2 mRNA expression, whereas TLR‐4 mRNA expression was not changed. P. gingivalis lipopolysaccharide induced interleukin‐6 and interleukin‐8 production in chorion‐derived cells, but the P. gingivalis lipopolysaccharide‐induced interleukin‐6 and interleukin‐8 production was reduced in TLR‐2 gene‐silenced chorion‐derived cells. Conclusion: Our results suggest that P. gingivalis can be detected in chorionic tissues of hospitalized high‐risk pregnant women, and that P. gingivalis lipopolysaccharide induces interleukin‐6 and interleukin‐8 production via TLR‐2 in chorion‐derived cells.     

Induction of interleukin (IL)-1β, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans

Trindade SC, Olczak T, Gomes‐Filho IS, Moura‐Costa LF, Cerqueira EMM, Galdino‐Neto M, Alves H, Carvalho‐Filho PC, Xavier MT, Meyer R. Induction of interleukin (IL)‐1β, IL‐10, IL‐8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans. J Periodont Res 2012; 47: 27–32. © 2011 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis, an anaerobic gram‐negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)‐1β, IL‐8 and IL‐10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. Material and Methods: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. Results: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL‐1β and IL‐8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL‐10 in the CP group. Higher levels of IL‐1β and IL‐10 were induced by HmuY than by other antigens derived from the wild‐type P. gingivalis strains. In contrast, total antigens derived from the hmuY‐deletion mutant strain induced the production of significantly higher levels of IL‐8 and significantly lower levels of IL‐1β. Conclusion: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host–pathogen interactions.     

Porphyromonas gingivalis promotes monocyte migration by activating MMP-9

Zhou J, Zhang J, Chao J. Porphyromonas gingivalis promotes monocyte migration by activating MMP‐9. J Periodont Res 2012; 47: 236–242. © 2011 John Wiley & Sons A/S Background and Objective: The migration of monocytes into the local environment is crucial for their maturation into macrophages or osteoclasts in the pathogenesis of periodontal disease. The objective of this study was to investigate the role and mechanisms mediated by Porphyromonas gingivalis in promoting the migration of monocytes by regulating MMP‐9 and TIMP‐1 expression. Material and Methods: Human THP1 monocytes were treated with culture supernatant derived from P. gingivalis (ATCC 33277) for 24 h. Zymography, western blot analysis and quantitative PCRs were performed to analyse protein and mRNA levels of MMP‐9. Protein and mRNA levels of TIMP‐1 from monocytes treated with or without P. gingivalis were determined as well. Transwell migration assay was carried out to analyse the effect of P. gingivalis on the migration of human peripheral blood CD14‐positive monocytes. An MMP inhibitor (GM6001) and a proteinase inhibitor (leupeptin) were used to determine the role of MMP‐9 in P. gingivalis supernatant‐ and lipopolysaccharide‐induced monocyte migration. Results: In zymography and western blot, an 82 kDa band of active MMP‐9 emerged in P. gingivalis‐treated monocyte culture media in a dose‐dependent manner, in addition to the MMP‐9 proenzyme (92 kDa) band expressed in control cell culture media. P. gingivalis supernatant increased both the protein and the mRNA levels of MMP‐9 and TIMP‐1. P. gingivalis supernatant, but not its lipopolysaccharide, increased the migratory ability of CD14‐positive monocytes. The increased migratory ability of P. gingivalis‐treated monocytes was partly inhibited by leupeptin (200 μg/mL) and completely antagonized by the MMP inhibitor GM6001 (100 nm ). Lipopolysaccharide of P. gingivalis increased protein and mRNA levels of MMP‐9 in monocytes, but had no effect on the migratory ability or MMP‐9 activation. Conclusion: P. gingivalis supernatant increased the migratory ability of monocytes, in part, by increasing activation and expression of MMP‐9.     

Oral infection with Porphyromonas gingivalis and systemic cytokine profile in C57BL/6.KOR-ApoE shl mice

Miyauchi S, Maekawa T, Aoki Y, Miyazawa H, Tabeta K, Nakajima T, Yamazaki K. Oral infection with Porphyromonas gingivalis and systemic cytokine profile in C57BL/6.KOR‐ApoE^shlmice. J Periodont Res 2012; 47: 402–408. © 2011 John Wiley & Sons A/S Background and Objective: Periodontal infection affects atherosclerotic diseases, such as coronary heart diseases. Mouse models have revealed that oral infection with Porphyromonas gingivalis induces changes in inflammatory‐ and lipid metabolism‐related gene expression, regardless of the development of atherosclerotic lesions. However, the serum protein expression profile in the oral infection model has not been investigated. The present study aimed to analyse the effect of oral infection with P. gingivalis on the expression levels of multiple cytokines in the serum in apolipoprotein E‐deficient mice by using a cytokine antibody array. Material and Methods: C57BL/6.KOR‐Apoe^shl mice were orally infected with P. gingivalis five times at 3 day intervals and were then killed. Splenocytes were isolated and analysed for proliferative activity and immunoglobulin G (IgG) production in response to in vitro restimulation with P. gingivalis. The expression levels of various cytokines in the sera were analysed using a mouse antibody array glass chip. Results: Splenocytes from P. gingivalis‐infected mice demonstrated significantly greater proliferation and IgG production in response to P. gingivalis compared with those from sham‐infected mice. Antibody array analysis revealed the selective upregulation of matrix metalloproteinase 3, intercellular adhesion molecule 1, insulin‐like growth factor binding protein 2 and chemokine (C‐X‐C motif) ligand 7 and the downregulation of interleukin‐17, tumor necrosis factor‐α and L‐selectin. Conclusion: These data demonstrate that oral infection with P. gingivalis induces alterations in systemic cytokine production. These cytokines could play roles in the development not only of periodontitis but also of atherosclerosis.     

Reduced expression of lipopolysaccharide‐induced CXC chemokine in Porphyromonas gingivalis‐induced experimental periodontitis in matrix metalloproteinase‐8 null mice

Hernández M, Gamonal J, Salo T, Tervahartiala T, Hukkanen M, Tjäderhane L, Sorsa T. Reduced expression of LIX/CXCL5 in Porphyromonas gingivalis‐induced experimental periodontitis in matrix metalloproteinase‐8 null mice. J Periodont Res 2011; 46: 58–66. © 2010 John Wiley & Sons A/S Background and Objective: Matrix metalloproteinase‐8 (MMP‐8) is a central mediator in chronic periodontitis. Recently developed MMP‐8‐deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis‐induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide‐induced CXC chemokine (LIX/CXCL5), stromal‐derived factor‐1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis‐induced experimental periodontitis in MMP‐8^?/^? (knockout) and wild‐type mice. Material and methods:  MMP‐8 null and WT P. gingivalis‐infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified. Results: Typical histopathological features of chronic periodontitis were seen in P. gingivalis‐infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild‐type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild‐type P. gingivalis‐infected groups compared with uninfected groups (p < 0.05). Conclusion: RANKL and SDF1/CXCL12 are up‐regulated in P. gingivalis‐induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP‐8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis‐induced experimental periodontitis model.     

Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase-2 induction

Aoyama N, Suzuki J, Wang D, Ogawa M, Kobayashi N, Hanatani T, Takeuchi Y, Izumi Y, Isobe M. Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase‐2 induction. J Periodont Res 2011; 46: 176–183. © 2010 John Wiley & Sons A/S Background and Objective: Abdominal aortic aneurysm (AAA) is a common and lethal disorder, and MMPs are highly expressed in AAA lesions. Large numbers of periodontopathic bacteria have been reported to be present in specimens obtained from the aortic walls of patients with an AAA. The purpose of this study was to analyze the influence of periodontopathic bacteria on AAA dilatation. Material and Methods: AAAs were produced in mice by the periaortic application of 0.25 m CaCl₂, and NaCl was used as a control. The mice were inoculated once weekly with live Porphyromonas gingivalis, live Aggregatibacter actinomycetemcomitans or vehicle. Results: Four weeks after the periaortic application of either CaCl₂ or NaCl, a significant increase was observed in the aortic diameter of P. gingivalis‐challenged mice compared with the vehicle control mice (p < 0.05), whereas there was no statistically significant increase in the aortic diameter of the A. actinomycetemcomitans‐challenged mice. Immunohistochemical analysis found significantly higher numbers of CD8‐positive and MOMA2‐positive cells and significantly higher levels of MMP‐2 in the aneurysmal samples of P. gingivalis‐challenged mice compared with control mice. Live P. gingivalis promoted a significant proliferation of splenocytes in comparison with P. gingivalis‐lipopolysaccharide and live A. actinomycetemcomitans (p < 0.05). Conclusion: These findings demonstrate that challenge with P. gingivalis, but not with A. actinomycetemcomitans, can accelerate, or even initiate, the progression of experimental AAA through the increased expression of MMPs.     

Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. Porphyromonas gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes, 40 and 43 kDa, in the cell. The 43‐kDa and 40‐kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40‐kDa Mfa3 alone. Furthermore, the 40‐kDa Mfa3 started with the Ala⁴⁴ residue of the deduced amino acid sequence, indicating that the N‐terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno‐electron microscopy revealed that Mfa3 localized at the tip of the fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in the absence of other accessory proteins, PGN0290 and PGN0291, in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto‐aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1^–, FimA^–). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Gingival fibroblast responsiveness is differentially affected by Porphyromonas gingivalis: implications for the pathogenesis of periodontitis

In periodontitis, tissue damage results mainly from aberrant host responses to oral microorganisms. Fibroblasts can play an important role in this. Gingival fibroblasts do not develop tolerance against the lipopolysaccharide of Porphyromonas gingivalis, a keystone pathogen in periodontitis, which may partly explain the persistence of inflammation. However, besides lipopolysaccharide, live P. gingivalis possess numerous virulence traits to impair host‐responses. We hypothesized that fibroblast‐responsiveness to a bacterial challenge could be affected by live P. gingivalis. We investigated if inflammatory responses of gingival fibroblasts to P. gingivalis were altered, when the fibroblasts had encountered P. gingivalis previously. On consecutive days, primary human gingival fibroblasts were challenged twice for 6 h with live P. gingivalis, or fibroblasts were preincubated for 24 h with a lower concentration of live P. gingivalis and re‐challenged for 6 h with a higher concentration. As the P. gingivalis capsule and proteases are involved in modulating host responses, we used encapsulated P. gingivalis W83 and a non‐encapsulated mutant, and P. gingivalis ATCC33277 and a lys‐gingipain and arg‐gingipain mutant, to challenge fibroblasts. With all P. gingivalis‐strains, interleukin‐8 and monocyte chemoattractant protein‐1 responses to the second challenge were less strong in fibroblasts that had been challenged with P. gingivalis before. These lower responses might correspond with higher interleukin‐1 receptor agonist expression. Fibroblast responses to a second challenge were not influenced by 24 h preincubation. Reduced chemokine responses after consecutive potent P. gingivalis challenges indicate that gingival fibroblast responsiveness is affected by a previous bacterial encounter. In periodontitis, such reduced chemokine responses may impair chemotaxis and clearance of oral microorganisms, thereby leading to prolonged inflammatory responses and tissue damage.     

Identification of functional domains of the minor fimbrial antigen involved in the interaction of Porphyromonas gingivalis with oral streptococci

Porphyromonas gingivalis is associated with chronic periodontitis and may initially colonize the oral cavity by adhering to streptococci. Adhesion to streptococci is driven by interaction of the minor fimbrial antigen (Mfa1) with streptococcal antigen I/II. We identified the region of antigen I/II required for this interaction and developed small molecule mimetics that inhibited P. gingivalis adherence. However, the functional motifs of Mfa1 involved in the interaction with antigen I/II remain uncharacterized. A series of N‐ and C‐terminal peptide fragments of Mfa1 were expressed and tested for inhibition of P. gingivalis adherence to S. gordonii. This approach identified residues 225–400 of Mfa1 as essential for P. gingivalis adherence. Using the three‐dimensional structure of Mfa1, a putative binding cleft was identified using SiteMap and five small molecule mimetics could dock in this site. Site‐specific mutation of residues in the predicted cleft, including R240A, W275A, D321A and A357P inhibited the interaction of Mfa1 with streptococci, whereas mutation of residues not in the predicted cleft (V238A, I252F and ΔK253) had no effect. Complementation of an Mfa1‐deficient P. gingivalis strain with wild‐type mfa1 restored adherence to streptococci, whereas complementation with full‐length mfa1 containing the R240A or A357P mutations did not restore adherence. The mutations did not affect polymerization of Mfa1, suggesting that the complemented strains produced intact minor fimbriae. These results identified specific residues and structural motifs required for the Mfa1‐antigen I/II interaction and will facilitate the design of small molecule therapeutics to prevent P. gingivalis colonization of the oral cavity.     

Interaction of oral bacteria with gingival epithelial cell multilayers

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air–liquid interface in low‐calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 secretion; and F. nucleatum stimulated production of IL‐1β and TNF‐α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.     

Divergence of the systemic immune response following oral infection with distinct strains of Porphyromonas gingivalis

Periodontitis is a polymicrobial oral infection characterized by the destruction of tooth‐supporting structures that can be linked to systemic diseases such as cardiovascular disease, diabetes or rheumatoid arthritis. Porphyromonas gingivalis, a bacterium implicated in the etiology of periodontitis, has shown variation in inducing T‐cell responses among different strains. Therefore, in this study we investigated the strain‐specific immune response using a murine experimental model of periodontitis. Periodontitis was induced by P. gingivalis strains A7A1‐28, W83 and W50, and later confirmed by the presence of P. gingivalis in the oral microflora and by alveolar bone resorption. Splenocytes were evaluated for gene expression, cellular proteins and cytokine expression. Dendritic cells were stimulated in vitro for T helper cell–cytokine profiling. Results showed that P. gingivalis had the ability to alter the systemic immune response after bacterial exposure. Strains W50 and W83 were shown to induce alveolar bone loss, whereas the A7A1‐28 strain did not significantly promote bone resorption in mice. Splenocytes derived from mice infected with strains W50 and W83 induced expression of high levels of interleukin‐4 (IL‐4) but A7A1‐28 stimulated increased IL‐10. Stimulation of dendritic cells in vitro showed a similar pattern of cytokine expression of IL‐12p40, IL‐6 and transforming growth factor‐β among strains. A distinct systemic response in vivo was observed among different strains of P. gingivalis, with IL‐10 associated with the least amount of alveolar bone loss. Evaluation of pathogen‐driven systemic immune responses associated with periodontal disease pathogenesis may assist in defining how periodontitis may impact other diseases.     

Identification of immunoreactive epitopes of the Porphyromonas gingivalis heat shock protein in periodontitis and atherosclerosis

Choi J, Lee S‐Y, Kim K, Choi B‐K. Identification of immunoreactive epitope of Porphyromonas gingivalis heat shock protein peptide in periodontitis and atherosclerosis. J Periodont Res 2011; 46: 240–245. © 2011 John Wiley & Sons A/S Background and Objective: Heat shock protein 60 (HSP60) of Porphyromonas gingivalis, a major periodontal pathogen, might be a trigger molecule linking infectious periodontitis and autoimmune atherosclerosis. The aim of this study was to identify the peptide specificity of anti‐P. gingivalis HSP60 monoclonal antibodies and their cross‐reactivity with bacterial and human HSPs. Their specific immunoreactivity to periodontal or atherosclerotic lesions was also investigated. Methods: Twenty patients with chronic periodontitis and 20 atherosclerosis patients who had undergone surgical intervention for atheromatous plaques with evidence of ongoing periodontal disease, were selected. Synthetic peptide 19 ((TLVVNRLRGSLKICAVKAPG)‐specific T‐cell lines were established from inflamed gingiva and atheromatous plaque and the phenotypes and cytokine profiles were characterized. Results: Thirty per cent of periodontitis patients and 100% of atherosclerosis patients reacted positively to cross‐reactive peptide 19 from both P. gingivalis and human HSP60. The peptide 19‐specific T‐cell lines demonstrated the phenotype characteristic of helper T cells (CD4⁺) but did not express CD25 or FOXP3. The interleukin‐10 levels were elevated significantly in the peptide 19 T‐cell line. Conclusion: Synthetic peptide 19 of P. gingivalis HSP60 is an immunoreactive epitope in the periodontitis–atherosclerosis axis.     

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.